help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-1577
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jasinska, A.
Right arrow Articles by Fazleabas, A. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jasinska, A.
Right arrow Articles by Fazleabas, A. T.
Endocrinology Vol. 147, No. 9 4112-4121
Copyright © 2006 by The Endocrine Society

Human Chorionic Gonadotropin and Decidualization in Vitro Inhibits Cytochalasin-D-Induced Apoptosis in Cultured Endometrial Stromal Fibroblasts

Anna Jasinska, Zuzana Strakova, Maciej Szmidt and Asgerally T. Fazleabas

Department of Obstetrics and Gynecology (A.J., Z.S., M.S., A.T.F.), University of Illinois, Chicago, Illinois 60612; and Department of Histology and Embryology (A.J., M.S.), Warsaw Agricultural University, 02 776 Warsaw, Poland

Address all correspondence and requests for reprints to: Asgi T. Fazleabas Ph.D., Department of Obstetrics and Gynecology University of Illinois, 820 South Wood Street, M/C 808, Chicago, Illinois 60612-7313. E-mail: asgi{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endometrial apoptosis increases from the proliferative phase through the secretory phase and peaks at menses. However, with the onset of pregnancy, the corpus luteum is rescued and stromal cells, instead of undergoing apoptosis, reorganize the cytoskeleton and then begin to differentiate. We hypothesized that in the presence of hormones (estradiol-17ß and medroxyprogesterone acetate), chorionic gonadotropin (hCG) as an early embryonic signal, and induction of decidualization by dibutyryl-cAMP (dbcAMP), endometrial stromal cells are rescued by the regulation of proteins that inhibit apoptosis. The percentage of cells stained with annexin V, an early apoptotic marker, increased dramatically after cytoskeletal disruption with cytochalasin D compared with non-cytochalasin-D-treated controls (P < 0.05). However, treatment of cells with hCG or dbcAMP in the presence of hormones significantly (P < 0.05) decreased the percentage of annexin-V-stained cells compared with cells treated with cytochalasin D alone. This inhibition was further confirmed by immunodetection of cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The inhibition of apoptosis by hCG and dbcAMP was via the intrinsic pathway because the cytochalasin-D-treated cells stained intensely for Bax, whereas the cells treated with hormones, hCG, or dbcAMP stained predominantly for Bcl-2. Treatment of cytochalasin-D-treated cells with hormones and dbcAMP resulted in an increase in the secretion of IGF-binding protein-1 (IGFBP-1) and prolactin. Treatment of cytochalasin-D-treated cells with recombinant IGFBP-1 and prolactin also inhibited apoptosis. These data suggest that under in vitro conditions, both hCG and the induction of decidualization play a direct role in preventing uterine stromal cells from undergoing apoptosis. Furthermore, this inhibition of apoptosis may be mediated in part by IGFBP-1 and prolactin and the alteration in the expression of Bcl-2 and Bax.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS IS A type of programmed cell death that plays an important role during embryonic development and tissue homoeostasis in the adult. Apoptosis has been shown to be an important regulator of endometrial function.

In the human endometrium, apoptosis was first described in 1975 by Hopwood and Levison (1), and it was later reported that the level of apoptosis increases from proliferative phase through the cycle and peaks at menses (2). However, our recent studies have indicated that treatment with human chorionic gonadotropin (hCG) or progesterone at the mid-secretory phase inhibits apoptosis in late secretory phase, implying a role for embryonic signals in the inhibition of apoptosis (3).

Apoptosis is regulated by various gene products including cytokines, interleukins, and steroid hormones. In addition, the cytoskeleton may also play a role in the early stages of induction of apoptosis (4). This is suggested by the finding that DNase I, an endonuclease shown to be responsible for nuclear fragmentation during apoptosis, is associated with full-length actin in live cells, and this association down-regulates its endonuclease activity (5). Cell shape is also governed by the cytoskeleton, which acts as a mechanical supporting framework (6) as well as an orienting foundation for much of the cell signal transduction machinery (7). More supporting evidence comes from recent work indicating that the disruption of the actin cytoskeleton by dissociating agents induces apoptosis in some cell types (8). In mammary epithelial cells, disruption of the connections between the actin cytoskeleton and the extracellular matrix (ECM) has deleterious effects on cell survival, because attachment to the extracellular matrix and the stimulation of growth factor signaling cascades suppressed the activity of the BH-3 family of apoptotic proteins (9). Furthermore, additional studies (10, 11) also demonstrated the importance of Fas to actin linkage and its role in Fas-mediated apoptosis in lymphocytes. These studies suggested that Fas linkage to actin may play a role in conferring cell susceptibility to Fas, triggering and driving the actin-dependent death-inducing signaling complex and the internalization of Fas (12).

Differentiation of stromal fibroblasts into the secretory decidual phenotype is initially associated with changes in the cytoskeleton (13). We previously reported that stromal fibroblasts express {alpha}-smooth muscle actin ({alpha}-SMA), a cytoskeletal protein in response to in vivo stimulation by hCG (14). It has been suggested that this induction of {alpha}-SMA occurs as a consequence of integrins on the stromal cell membrane binding to secreted ECM proteins (15). The interaction between integrins and the ECM induces changes in the actin cytoskeleton that are thought to be critical for signal transduction (16). After implantation, additional signals from the embryo initiate the process of decidualization at the interface between the maternal endometrium and the embryo (17, 18, 19). This differentiation process is associated with the down-regulation of {alpha}-SMA and the up-regulation of IGF-binding protein-1 (IGFBP-1). Continued exposure to hCG in the absence of a conceptus results in the prolonged expression of {alpha}-SMA in stromal fibroblasts, and these cells do not transform into the decidual phenotype (17). However, in the presence of a conceptus or after decidualization in vitro, the stromal fibroblasts decrease their expression of {alpha}-SMA and up-regulate their expression of IGFBP-1 (19, 20). Furthermore, disruption of the actin filaments by cytochalasin D, a specific inhibitor of actin polymerization, followed by treatment with steroid hormones and dibutyryl-cAMP (dbcAMP) promotes rapid stromal cell differentiation and decidualization (13). Cytochalasin D caps the barbed or rapidly growing ends of actin filaments and prevents the addition of G-actin monomers (21, 22). This causes complete disruption of actin filament integrity by creating short filaments and aggregates that function at a reduced capacity (23). Treatment of confluent stromal cells with 10 µM cytochalasin D in serum-free medium for 24 h causes the derangement in actin filament structure (13). Cells lose their fibroblast-like shape and become rounded with numerous thin filopodia and respond rapidly to the cAMP stimulus (13).

Because our previous in vivo studies had demonstrated that hCG and progesterone inhibited endometrial apoptosis in the late secretory phase (3), this study examined the mechanisms by which hCG rescues stromal cell apoptosis after cytoskeletal disruption. Furthermore, because it has also been reported that IGFBP-1 can inhibit Fas-induced hepatic apoptosis in the IGFBP-1 null mouse (24) and that prolactin, another decidual protein, inhibits decidual apoptosis in the rodent (25), a second aim of the study was to determine whether the induction of IGFBP-1 and prolactin by hormones and dbcAMP after cytoskeleton disruption (13, 20) is also associated with the inhibition of apoptosis of human stromal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant hCG was obtained from Dr. A. F. Parlow (National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA) and Serono Pharmaceuticals Inc. (Randolph, MA). A monoclonal antibody against cleaved caspase-3 was from Cell Signaling Technology, Inc. (Beverly, MA). Monoclonal ß-actin antibody clone AC 15; N6, 2'-O-dibutyryladenosine 3,5'-cyclic monophosphate (dbcAMP) and cytochalasin D were from Sigma Chemical Co. (St. Louis, MO). Annexin V-fluorescein isothiocyanate (FITC) and a monoclonal antibody against Bcl-2 and Bax was from BD Bioscience (San Diego, CA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis detection kit (ApoptoTag Plus Peroxidase) was from Chemicon International (Temecula, CA). All cell culture supplies were obtained from Life Technologies, Inc. (Gaithersburg, MD). Other reagents of cell culture grade were purchased from Fisher Scientific (Itasca, IL), Sigma, or Roche Molecular Biochemicals (Indianapolis, IN).

Cell isolation
Human uterine stromal fibroblasts were isolated from the decidua parietalis dissected from the placental membranes after normal vaginal delivery at term. Previous studies from our laboratory (20) and others (26, 27) have shown that these stromal fibroblasts respond to hormonal stimulation in a manner that is comparable to stromal cells isolated from the uterine endometrium during the menstrual cycle. These studies were approved by the Institutional Review Board of the University of Illinois.

Stromal cell isolation was performed as previously described (13, 20). Cells were seeded at 1 x 105 cells/cm2 and placed into an incubator at 37 C, 5% CO2. The next day, the plates were extensively washed with PBS to remove nonadherent (mainly decidual) cells. At confluence, cells were trypsinized and used for experiments in passage numbers 3–5. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (Dako Corp., Carpinteria, CA) and vimentin (Zymed Laboratories, Inc., San Francisco, CA). The purity of the stromal cell preparations used in these studies was greater than 97%.

Treatment of cells
At approximately 80% confluence, the cell culture medium for stromal cells was changed to serum-free RPMI 1640 supplemented with sodium pyruvate, penicillin/streptomycin, and with or without 10 µM cytochalasin D. After the 24-h pretreatment with cytochalasin D, medium was exchanged, and the cells were left untreated or treated with hormones [36 nM estradiol-17ß and 1 µM medroxyprogesterone acetate (MPA)], with hormones and 0.1 mM dbcAMP, with 50 IU hCG, or with hormones and hCG for an additional 24 h. All experiments were repeated a minimum of three times, and each experiment was done in duplicate.

Flow cytometry analysis
For flow cytometry analysis, cells were prepared after the protocol of annexin V-FITC/propidium iodide (PI) assay (BD PharMingen). The stromal cells were detached by 0.25 g/liter trypsin, 0.1 g/liter EDTA treatment and washed twice with ice-cold PBS. Supernatants were removed, and cell pellets were resuspended in 100 µl of ice-cold binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2]. Five microliters of annexin V-FITC and 10 µl of 50 µg/ml PI were added to the cell suspension of each sample. Tubes were then incubated for 15 min at room temperature without exposure to light. After placement on ice, 400 µl of binding buffer was added into each sample before analysis by flow cytometry. Ten thousand events were analyzed per test using Becton Dickinson (Franklin Lakes, NJ) FACSCalibur Flow Cytometer. The results are expressed as the percentage of apoptotic annexin V-FITC-positive cells of total cells counted.

TUNEL apoptosis detection assay
Stromal cells were grown on the eight-well chamber slides. After the treatment, media were removed and the cells were washed three times with PBS. Cells were fixed in 1% paraformaldehyde for 10 min at room temperature and washed three times in PBS. The cells were then permeabilized in ethanol:acetic acid (2:1) and washed in PBS. Slides were covered with TUNEL solution (TdT and digoxigenin-dUTP) and were incubated in humidified atmosphere at 37 C for 60 min. The reaction was terminated by rinsing with PBS. Antidigoxigenin antibody conjugated with peroxidase was added to the samples. To visualize the immunocomplexed peroxidase, substrate solution (75 µl) was added. The cells were counterstained with methyl green. The experiment was repeated three times in duplicate. TUNEL staining was quantitated by counting the number of positively stained stromal cells per hundred cells within 10 different high-power (x40) fields for each treatment group. Values represent the mean percentage of positively stained cells ± SD.

Preparation of cell lysates and immunodetection
After each treatment, stromal cells were rinsed twice with ice-cold PBS and lysed on ice with lysis buffer, as previously described (20). Equal amounts of total protein (10 µg) were separated by 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking for 1 h in PBS-Tris containing 5% nonfat dry milk, the blots were then incubated overnight with antibodies against cleaved caspase-3 (1:1000, no. 9664; Cell Signaling Technology). After incubation for 1 h with the secondary antirabbit IgG labeled with horseradish peroxidase (1:3000; Amersham Pharmacia Biotech, Piscataway, NJ), immunocomplexes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). The blots were subsequently stripped and incubated with antibodies against ß-actin (1:5000; Sigma AC-15) as a loading control.

Immunofluorescent staining
Human uterine fibroblasts were grown on glass coverslips. Cells were pretreated with cytochalasin D followed by incubation in the presence of hormones, hCG, and dbcAMP for 24 h, as described above. Control cells were left untreated. Cells were fixed in 4% paraformaldehyde for 10 min and washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100/0.1% deoxycholate in PBS for 10 min. A monoclonal Bcl-2 or Bax antibody (1:1000 in 1% BSA in PBS) was added to the cells and incubated overnight at 4 C. Cells were washed three times with PBS and incubated with a fluorescein-conjugated antimouse IgG second antibody (10 µg/ml; Vector Laboratories, Inc., Burlingame, CA). Fluorescence corresponding to the antigen-antibody complexes for Bcl-2 and Bax were visualized with the Nikon Eclipse E400 series fluorescent microscope (Fryer Co., Huntley, IL). Images were captured using a digital Spot Camera and the Image-Pro Plus software package (Media Cybernetics, San Diego, CA).

The results of immunostaining for Bcl-2 and Bax were examined semiquantitatively by using an immunohistochemical histological score (H-score) that incorporates both the intensity and the distribution of specific staining. The H-score has been previously formulated as HS = {Sigma}({pi}x i)/100, where {pi} denotes the percentage of stained cells and i denotes the intensity of the staining ranging from 1–3 (28). Three different high-power fields (x40) were examined. Both staining intensity and number of positive cells were counted. In evaluating the staining intensity, a score of zero indicated an absence of staining. Scores of 1, 2, and 3 indicated weak, moderate, and strong immunoreactivity, respectively.

Detection of the IGFBP-1 and prolactin
After each of the treatments, IGFBP-1 and prolactin levels in the culture medium were measured using an ELISA kit (Diagnostic Systems Laboratories, Inc., Webster, TX) as previously described (19). The concentration of each of the decidualization markers in the conditioned media were normalized to the total amount of protein in each of the culture experiments.

Statistical analysis
Statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL). Means of flow cytometry data and H-scores for Bcl-2 and Bax staining were analyzed by one-way ANOVA at a significance level of P < 0.05. When the overall ANOVA was significant, post hoc tests were performed using Tukey’s correction for multiple comparisons. To determine whether there was a difference in the mean percentage of TUNEL-positive cells by treatment, the Kruskal-Wallis one-way ANOVA was performed. Pair-wise post hoc analysis of groups was done using the t test. The Bonferroni correction was used to determine the appropriate level of statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytoskeletal disruption induces human stromal cell apoptosis
We used three different analyses to confirm that the disruption of the actin cytoskeleton in stromal fibroblasts with cytochalasin D is correlated with the induction of apoptosis.

First, human stromal cells were stained with annexin V-FITC to examine the inversion of phosphatidylserine to the outer leaflet of the cell membrane (29). As shown in Fig. 1Go, after 24 h of treatment with cytochalasin D, there is a significant increase in annexin-V-positive staining compared with the untreated control. In the presence of cytochalasin D, approximately 50% of the cells stain positively for annexin V. To test our hypothesis that an embryonic signal can rescue stromal cells from undergoing apoptosis, cytochalasin-D-treated cells were rinsed with PBS and then incubated for an additional 24 h with estradiol-17ß and MPA in the presence or absence of hCG or with dbcAMP as the decidualization stimulus to mimic the events that occur in the endometrium during implantation. Addition of estradiol-17ß and MPA alone significantly (P < 0.05) decreased the number of cells staining positive for annexin V. This inhibition of apoptosis after cytoskeletal disruption was further enhanced with the cells treated with hCG alone or with hCG or dbcAMP in the presence of both estradiol and MPA.


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		FIG. 1. Regulation of apoptosis in human stromal cells by hCG and dbcAMP in presence of steroid hormones. A, Representative flow cytometric analysis of annexin V-FITC staining of human stromal cells after cytoskeletal disruption and treatment with hormones and dbcAMP; B, bar graph summarizing mean ± SD of three independent experiments in which each treatment was done in triplicate. Note the significant increase (P < 0.05) in the number of apoptotic cells after cytoskeleton disruption by cytochalasin D compared with the untreated cells and the significant rescue from apoptosis after the treatment with hormones and dbcAMP or hormones and hCG. *, Significant difference between cytochalasin D treatment and other treatment groups (P < 0.05). There were no significant differences in the number of cells staining for annexin V between each of the other treatment groups. cAMP, dbcAMP; CD, cytochalasin D; H, hormones (estradiol-17ß and MPA).

 
The flow cytometry data were further confirmed by TUNEL staining (Fig. 2Go). After disruption of the cytoskeleton caused by cytochalasin D, evidence for TUNEL staining in the cell nuclei was strong (Fig. 2BGo). Treatment with steroid hormones (Fig. 2CGo) or steroid hormones in combination with dbcAMP (Fig. 2DGo) or hCG (Fig. 2Go, E and F) reduced the intensity of TUNEL staining in these cells. Quantification of the TUNEL staining confirmed that these treatments resulted in a significant (P < 0.001) decrease in the number of cells that stained positive (Fig. 2BGo).


Figure 2
View larger version (98K):
[in this window]
[in a new window]
  		FIG. 2. Immunohistochemical staining for apoptotic cells detected by the TUNEL method. A, Near-confluent human stromal cells were treated with (B–F) or without (A) cytochalasin D for 24 h. Fresh serum-free RPMI 1640 with hormones (C), with hormones and dbcAMP (D), with hCG (E), or with hormones and hCG (F) was added to the cells for an additional 24 h. Note the decrease of TUNEL staining in the cells treated with hormones only (C) or with hormones and dbcAMP (D) compared with the cells left untreated after cytochalasin D treatment (B). Images are at the same magnification; the scale mark is on the image shown in A. B, Quantitative assessment of TUNEL staining. Cytochalasin-D-treated cells stained significantly (*, P < 0.001) more compared with controls and the treated cells after cytoskeletal disruption. cAMP, dbcAMP; CD, cytochalasin D; H, hormones (estradiol-17ß and MPA).

 
Caspase-3 activates endonuclease activity, thereby linking the cytosolic proteolytic activity to induce DNA fragmentation (30, 31, 32). Our previous studies (3) demonstrated that both hCG and progesterone could inhibit endometrial DNA degradation. This led us to examine whether cleaved and presumably active caspase-3 would be increased in stromal fibroblasts. As shown in Fig. 3Go, the active form of caspase-3 was most highly expressed in the cells that were treated with cytochalasin D. Treatment with estradiol-17ß and MPA or hCG alone decreased the amount of active caspase-3, which was undetectable when the cells were treated with estradiol-17ß and MPA together with dbcAMP or almost undetectable after hCG and hormones treatment.


Figure 3
View larger version (49K):
[in this window]
[in a new window]
  		FIG. 3. The expression of the active (cleaved) and nonactive (uncleaved) form of caspase-3 protein. Treatment with cytochalasin D (CD) resulted in an intense band of cleaved caspase-3. The amount of cleaved caspase-3 in the presence of hormones (H) and dbcAMP (cAMP) or hormones and hCG was significantly reduced. The blots were reprobed with ß-actin as loading control.

 
IGFBP-1 and prolactin inhibit stromal cell apoptosis
We previously reported that disruption of the actin filaments by cytochalasin D followed by the treatment with hormones and dbcAMP will rapidly induce stromal cell decidualization (13). To further confirm these data, the concentration of the IGFBP-1 and prolactin, as markers of decidualization, were measured in the conditioned media collected from the treated samples. As is shown in Fig. 4Go, only treatment with hormones and dbcAMP resulted in the secretion of both IGFBP-1 (mean, 130 pg/µg protein) and prolactin (mean, 2.8 pg/µg protein) into the medium. Neither hormones alone nor hCG with or without hormones induced the secretion of IGFBP-1 or prolactin by human stromal cells within 24 h after cytoskeletal disruption compared with controls.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Concentration of IGFBP-1 and prolactin in the conditioned medium. Medium was collected after 24 h of cytochalasin D (CD) pretreatment followed by hormones (H) and dbcAMP (cAMP) or hormones and hCG treatment for an additional 24 h. IGFBP-1 and prolactin protein released into the medium was measured using an ELISA. Data are expressed as least-squares means ± SEM of three experiments. Only treatment with hormones and dbcAMP after treatment with cytochalasin D resulted in secretion of both IGFBP-1 and prolactin proteins into the medium (P < 0.05).

 
Because both IGFBP-1 and prolactin have been shown to inhibit apoptosis in the rodent (24, 25), we wondered whether IGFBP-1 or prolactin could also rescue human stromal cells from undergoing apoptosis. To test this hypothesis, stromal cells disrupted with cytochalasin D were treated with hormones or dbcAMP and hormones or with recombinant IGFBP-1 (50 and 200 ng; Upstate, Lake Placid, NY) or prolactin (10 and 100 ng; Cell Sciences, Inc., Canton, MA) for 24 h. The cells were then analyzed for annexin V staining as described above. As shown in Fig. 5Go, the flow cytometry analysis demonstrated that the addition of either IGFBP-1 or prolactin inhibited stromal cell apoptosis at levels that were comparable to those observed in the presence of hormones and dbcAMP.


Figure 5
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Inhibition of apoptosis by IGFBP-1 and prolactin in human stromal cells. The percentage of apoptotic cells was evaluated by flow cytometry using annexin V-FITC. The results represent the mean ± SD from three independent experiments done in triplicate. *, Values statistically different from the control and treated cells (P < 0.05). Note the decrease in the number of apoptotic cells in the presence of recombinant IGFBP-1 (BP-1) as well as prolactin (PRL) at both doses. The level of inhibition of apoptosis by IGFBP-1 and prolactin is comparable to the level observed in the samples treated with steroid hormones (H) and dbcAMP (cAMP). CD, Cytochalasin D.

 
In our previous in vivo study (3), inhibition of late secretory phase apoptosis by hCG and progesterone was associated with an increase in antiapoptotic protein Bcl-2 and a corresponding decrease of the proapoptotic protein Bax. Similar to our in vivo results, immunofluorescent staining with Bax antibody showed a similar pattern as TUNEL staining. Bcl-2 protein expression was detected at a high level in untreated controls. Bcl-2 protein expression was detected at a high level in untreated controls possibly because of the proliferative nature of these cells (Fig. 6AGo). A decrease in fluorescent staining was observed in samples treated with cytochalasin D (Fig. 6BGo). Addition of the steroid hormones alone (Fig. 6CGo) or in combination with dbcAMP (Fig. 6DGo) resulted in an increase of Bcl-2 staining compared with the cells treated with cytochalasin D only (Fig. 6BGo). Similarly, cells treated with hCG (Fig. 6EGo) or hCG and hormones (Fig. 6FGo) also resulted in an increase in Bcl-2 staining in the stromal cells. However, the intensity of staining was much less than cells treated with steroid hormones alone or those undergoing decidualization. Proapoptotic Bax proteins were highly expressed in samples treated with cytochalasin D (Fig. 7BGo). The intensity of fluorescent staining decreased when samples were treated with steroid hormones (Fig. 7CGo) and was further decreased with hCG (Fig. 7EGo) or steroid hormones in combination with either dbcAMP (Fig. 7DGo) or hCG (Fig 7FGo).


Figure 6
View larger version (43K):
[in this window]
[in a new window]
  		FIG. 6. Immunofluorescent localization of Bcl-2. Near-confluent human uterine fibroblasts were pretreated with (B–H) or without (A) cytochalasin D for 24 h. Fresh serum-free RPMI 1640 medium containing hormones (C), hormones and dbcAMP (D), hCG (E), hormones and hCG (F), 200 ng recombinant IGFBP-1 (G), or 100 ng recombinant prolactin (H) was added to the cells for another 24 h. Note the visible decrease in Bcl-2 staining in the cells treated with cytochalasin D (B) compared with control (A) and other treatment groups (C–H). Images are at the same magnification; the scale mark is on the image shown in A.

 

Figure 7
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 7. Immunofluorescent localization of Bax. Human uterine stromal cells were pretreated with (B–H) or without (A) cytochalasin D for 24 h after an additional 24 h of treatment with hormones (C), hormones and dbcAMP (D), hCG (E), hormones and hCG (F), 200 ng recombinant IGFBP-1 (G), or 100 ng recombinant prolactin (H) in serum-free RPMI 1640 medium. Note the visible increase in Bax staining in the cells treated with cytochalasin D (B) compared with untreated control cells (A). Treatment of cells with each of the other specific treatments that inhibit apoptosis resulted in a decrease of Bax staining (C–H). Images are at the same magnification; the scale mark is on the image shown in A.

 
The decrease in annexin V staining in the presence of IGFBP-1 and prolactin (Fig. 5Go) was also reflected in staining patterns observed for Bcl-2 and Bax. Treatment with 200 ng recombinant IGFBP-1 or 100 ng prolactin after pretreatment with cytochalasin D increased the intensity of Bcl-2 staining (Fig. 6Go, G and H) to that comparable to the hCG-treated cells and decreased the intensity of Bax staining to control levels (Fig. 7Go, G and H) when compared with the samples treated with cytochalasin D only (Figs. 6BGo and 7BGo). Quantification of the staining intensity for Bcl-2 and Bax by H-score are summarized in Fig. 8Go.


Figure 8
View larger version (22K):
[in this window]
[in a new window]
  		FIG. 8. H-score analysis of Bcl-2 and Bax staining. A, Bar graph representing H-score evaluation of staining intensity of Bcl-2; B, bar graph representing H-score evaluation of staining intensity of Bax. *, Values statistically different from the control and treated cells (P < 0.05). BP-1, Recombinant IGFBP-1 (200 ng); cAMP, dbcAMP; CD, cytochalasin D; H, hormones (estradiol-17ß and MPA); PRL, recombinant prolactin (100 ng).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis is a physiological process of cell death that is characterized by both morphological and biochemical events that results in the selective loss of cells within tissues without eliciting an inflammatory response. In the human endometrium, apoptosis is primarily evident in the glandular epithelium of the late secretory phase and in the menstruating endometrium (2, 33, 34). However, to maintain a pregnancy and facilitate the implantation process, it is critical that the endometrium be protected from undergoing apoptosis in the secretory phase. The sequence of biochemical and molecular events associated with this process still remains unclear. One of the possible factors that may help to prevent apoptosis is hCG secreted by the trophoblast cells. hCG causes the rescue of corpus luteum, and factors produced by corpus luteum such as progesterone help to maintain the pregnancy and protect the late luteal endometrium from undergoing apoptosis (3). It is also conceivable that hCG may directly act on the endometrium to inhibit apoptosis. Indeed, based on TUNEL staining, it appeared that hCG was more effective in reducing this marker of apoptosis in vivo when compared directly with progesterone supplementation (3).

We have previously shown that hCG has a direct effect on the endometrial stromal cells and modulates the cytoskeleton (14). It is known that the cytoskeleton plays a critical role in mitosis, cell growth, cell motility, and inhibition of apoptosis (35, 36).

To test the hypothesis whether hCG has a direct effect on preventing endometrial stromal cells from undergoing apoptosis, we undertook a series of in vitro experiments to determine the importance of the cytoskeletal reorganization in preventing stromal fibroblasts from undergoing apoptosis after implantation. Disruption of the cytoskeleton resulted in a dramatic increase in the number of cells undergoing apoptosis. Addition of estradiol-17ß and MPA partially inhibited this process. However, the addition of either hCG or dbcAMP together with steroids had a very significant effect on inhibiting the apoptotic process. The artificial disruption of the cytoskeleton with cytochalasin D differs from the physiological response of IL-1ß in reorganizing the cytoskeleton in vivo during the window of implantation (19). However, the effects of hCG and steroids on preventing stromal cell apoptosis correspond to our previous in vivo studies (3), which also resulted in a marked decrease in TUNEL staining and Bax localization with a corresponding increase in Bcl-2 staining. In vivo infusion of hCG into the uterine lumen of baboons during the window of receptivity (14) decreases the secretion of proapoptotic factors such as Fas and TNF receptor type 1 (19). Furthermore, in cultured human granulosa cells, hCG increased the expression of survivin, an antiapoptotic gene that may play a role in supporting luteal function after the establishment of pregnancy (37). In contrast, Kayisli et al. (38) reported that hCG increases apoptosis in endometrial cells. However, these investigators used pharmacological concentrations of a urinary preparation of hCG, which may account for the discrepancy between the two studies. The mechanism by which hCG rescues the stromal cells from undergoing apoptosis in vitro after cytoskeletal disruption remains to be clarified. We postulate that Bcl-2 may play a functional role in this process. Bcl-2 serves a protective antiapoptotic function that counteracts the proapoptotic actions of Bax and Bcl-x (34). In the human endometrium, it is the ratio of Bax and Bcl-2 that regulates apoptosis (2, 39). Our recent in vivo study suggested a reciprocal expression of Bcl-2 and Bax in women treated with either hCG or progesterone (3), which was also confirmed in the present study. The ratio of Bcl-2 to Bax expression is a critical determination of cell fate. Elevated levels of Bcl-2 favors cell survival, whereas increased levels of Bax accelerate cell death by controlling mitochondrial permeability. In response to an apoptotic stimulus, Bax levels increase and homodimerization results in the release of cytochrome c (40) and the activation of caspases 9 and 3 (41).

Another mechanism by which hCG may inhibit stromal cell apoptosis may involve regulation of the protein Notch-1, a receptor important in development and cancer (42). Treatment of stromal cells in vitro with hCG induces the expression of Notch-1, which is enhanced in the presence of estradiol and progesterone (43). Recent studies have suggested that Notch-1 signaling regulates apoptosis, and the active forms of Notch-1 proteins are antiapoptotic (44, 45). Thus, our observations suggest that hCG can act on stromal fibroblasts to inhibit the cytochalasin-D-induced apoptosis. The mechanism by which hCG regulates this process appears to be by up-regulating the expression of antiapoptotic genes such as Bcl-2 and Notch-1. It is also interesting to note that the targeted disruption of the LH/CG receptor results in decreased gene expression in an apoptotic cluster of genes in the uterus (46).

In the rodent, paracrine or autocrine mechanisms may control apoptosis in specific regions of the decidua. Recent studies suggest that Bcl-2 and Bcl-xL promote the survival of the mesometrial decidua until fully functional, after which Bax becomes predominant, resulting in the regression of the decidua (47).

Our second series of studies demonstrated that the rapid induction of decidualization by treatment with steroids and dbcAMP is also associated with the inhibition of apoptosis.

In rodents, IGFBP-1 functions as a critical hepatic survival factor in the liver by reducing the level of proapoptotic signals and the suppression of matrix metalloproteinase-9 and TGFß-1 (24). The role of prolactin as an epithelial cell survival factor has been well established, and it has been proposed that prolactin and GH play an essential role in cell survival (48). Prolactin plays also an important antiapoptotic role in the decidua of pseudopregnant rats under in vitro conditions. Prolactin can inhibit both caspase-3 and activin A in primary and decidual cell lines and prevent these cells from undergoing apoptosis (25, 49). Based on the fact that the primate decidualized stromal cells secrete both IGFBP-1 and prolactin (20, 50, 51) (Fig. 4Go), this raised the question of whether these proteins play a similar role in primates and in humans as they do in rodents. The present study demonstrates that when human stromal cells were incubated with different concentrations of recombinant IGFBP-1 and prolactin, there was a significant inhibition of apoptosis in these cells, comparable to that seen with the steroid hormones and dbcAMP treatment. In addition, cells treated with both IGFBP-1 and prolactin also demonstrated an increase in Bcl-2 staining and decrease in Bax staining.

It is evident that the levels of cAMP are critical in preventing human stromal cells from undergoing apoptosis. When decidualization was inhibited in the presence of a cannabinoid receptor agonist, a marked decrease in cAMP was accompanied by a progressive increase in TUNEL staining and caspase-3 (52). Furthermore, these cells also had a decreased expression of IGFBP-1 together with other decidualization markers, implying an important role for the decidualization response in the prevention of apoptosis. In addition, because prolactin in the rodent has been shown to inhibit caspase-3 (25), it is conceivable that the hormone plays a similar role in the human. However, the mechanisms by which IGFBP-1 inhibits apoptosis remain to be clarified. One possible mechanism may be associated with its ability to bind IGF-I, a well established survival factor (53). Alternatively, it may involve the interaction of the RGD domain on IGFBP-1 with cell adhesion molecules, which may compensate for the dissociation of the interaction of the cell adhesion molecules with the ECM as the cells round up after cytoskeletal dissociation (54, 55).

In summary, our studies suggest that both hCG and dbcAMP can prevent endometrial stromal fibroblasts from undergoing apoptosis after cytoskeletal disruption in vitro. We propose that initial change in the cytoskeletal architecture in response to hCG together with the induction of Notch-1 may be the early signal required to prevent the normal apoptotic cascade that is initiated toward the latter part of the menstrual cycle. In the presence of an embryo, decidualization is initiated, and this transformation requires the disruption or down-regulation of {alpha}-SMA (13, 18, 20). Thus, we hypothesize that the increase in IGFBP-1 and prolactin in decidual cells coupled with the down-regulation of the hCG receptor (56) compensates for the disruption of the cytoskeleton and the loss of hCG signaling and prevents the decidual cells from undergoing apoptosis as pregnancy proceeds.


   Footnotes
 
This work was supported by National Institues of Health Grant HD 42280 (A.T.F.).

Disclosure summary: A.J., Z.S., M.S., and A.T.F. have nothing to declare.

First Published Online June 1, 2006

Abbreviations: dbcAMP, Dibutyryl-cAMP; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; hCG, human chorionic gonadotropin; IGFBP-1, IGF-binding protein-1; MPA, medroxyprogesterone acetate; PI, propidium iodide; {alpha}-SMA, {alpha}-smooth muscle actin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Received December 13, 2005.

Accepted for publication May 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hopwood D, Levison DH 1978 Atrophy and apoptosis in the cyclical human endometrium. J Pathol 119:159–166
  2. Vaskivuo TE, Stenback F, Karhumaa P, Risteli J, Dunkel L, Tapanainen JS 2000 Apoptosis and apoptosis-related proteins in human endometrium. Mol Cell Endocrinol 165:75–83[CrossRef][Medline]
  3. Lovely LP, Fazleabas AT, Fritz MA, McAdams DG, Lessey BA 2005 Prevention of endometrial apoptosis: randomized prospective comparison of human chorionic gonadotropin versus progesterone treatment in the luteal phase. J Clin Endocrinol Metab 90:2351–2356[Abstract/Free Full Text]
  4. Suria H, Chau LA, Negrou E, Kelvin DJ, Madrenas J 1999 Cytoskeletal disruption induces T cell apoptosis by a caspase-3 mediated mechanism. Life Sci 65:2697–2707[CrossRef][Medline]
  5. Kayalar C, Ord T, Testa MP, Zhong LT, Bredesen DE 1996 Cleavage of actin by interleukin 1ß-converting enzyme to reverse DNase I inhibition. Proc Natl Acad Sci USA 93:2234–2238[Abstract/Free Full Text]
  6. Ingber DE 1993 Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci 104:613–627[Medline]
  7. Plopper GE, McNamee HP, Dike LE, Bojanowski K, Ingber DE 1995 Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol Biol Cell 10:1349–1365
  8. Kim SJ, Hwang SG, Kim IC, Chun JS 2003 Actin cytoskeletal architecture regulates nitric oxide-induced apoptosis, dedifferentiation, and cyclooxygenase-2 expression in articular chondrocytes via mitogen-activated protein kinase and protein kinase C pathways. J Biol Chem 278:42448–42456[Abstract/Free Full Text]
  9. Reddig PJ, Juliano RL 2005 Clinging to life: cell to matrix adhesion and cell survival. Cancer Metastasis Rev 24:425–439[CrossRef][Medline]
  10. Parlato S, Giammarioli AM, Logozzi M, Lozupone F, Matarrese P, Luciani F, Falchi M, Malorni W, Fais S 2000 CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway. EMBO J 19:5123–5134[CrossRef][Medline]
  11. Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME 2002 Molecular ordering of the initial signaling events of CD95. Mol Cell Biol 22:207–220[Abstract/Free Full Text]
  12. Fais S, De Milito A, Lozupone F 2005 The role of FAS to ezrin association in FAS-mediated apoptosis. Apoptosis 10:941–947[CrossRef][Medline]
  13. Kim JJ, Jaffe RC, Fazleabas AT 1999 Insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells: regulation by filamentous actin and requirement for de novo protein synthesis. Endocrinology 140:997–1004[Abstract/Free Full Text]
  14. Fazleabas AT, Donnelly KM, Srinivasan S, Fortman JD, Miller JB 1999 Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity. Proc Natl Acad Sci USA 96:2543–2548[Abstract/Free Full Text]
  15. Fazleabas AT, Bell SC, Fleming S, Sun J, Lessey BA 1997 Distribution of integrins and the extracellular matrix proteins in the baboon endometrium during the menstrual cycle and early pregnancy. Biol Reprod 56:348–356[Abstract]
  16. Clark EA, Brugge JS 1995 Integrins and signal transduction pathways: the road taken. Science 268:233–239[Abstract/Free Full Text]
  17. Christensen S, Verhage HG, Nowak G, de Lanerolle P, Fleming S, Bell SC, Fazleabas AT, Hild-Petito S 1995 Smooth muscle myosin II and {alpha} smooth muscle actin expression in the baboon (Papio anubis) uterus is associated with glandular secretory activity and stromal cell transformation. Biol Reprod 53:596–606
  18. Jasinska A, Han V, Fazleabas AT, Kim JJ 2004 Induction of insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells by cells of trophoblast origin. J Soc Gynecol Investig 11:399–405[Medline]
  19. Strakova Z, Mavrogianis P, Meng X, Hastings JM, Jackson KS, Cameo P, Brudney A, Knight O, Fazleabas AT 2005 In vivo infusion of interleukin-1ß and chorionic gonadotropin induces endometrial changes that mimic early pregnancy events in the baboon. Endocrinology 146:4097–4104[Abstract/Free Full Text]
  20. Strakova Z, Srisuparp S, Fazleabas AT 2000 Interleukin-1ß induces the expression of insulin-like growth factor binding protein-1 during decidualization in the primate. Endocrinology 141:4664–4670[Abstract/Free Full Text]
  21. Casella JF, Flanagan MD, Lin S 1981 Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature 293:302–305[CrossRef][Medline]
  22. Verrey F, Groscurth P, Bolliger U 1995 Cytoskeletal disruption in A6 kidney cells: impact on endo/exocytosis and NaCl transport regulation by antidiuretic hormone. J Membr Biol 145:193–204[Medline]
  23. Cooper JA 1987 Effects of cytochalasin and phalloidin on actin. J Cell Biol 105:1473–1478[Free Full Text]
  24. Leu JI, Crissey MA, Taub R 2003 Massive hepatic apoptosis associated with TGF-ß1 activation after Fas ligand treatment of IGF binding protein-1-deficient mice. J Clin Invest 111:129–139[CrossRef][Medline]
  25. Tessier C, Prigent-Tessier A, Ferguson-Gottschall S, Gu Y, Gibori G 2001 Prolactin antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity. Endocrinology 142:4086–4094[Abstract/Free Full Text]
  26. Richards RA, Brar AK, Frank GR, Hartman SM, Jikihara H 1995 Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression. Biol Reprod 52:609–615[Abstract]
  27. Brar AK, Handwerger S, Kessler CA, Aronow BJ 2001 Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization. Physiol Genomics 7:135–148[Abstract/Free Full Text]
  28. Huang A, Pettigrew NM, Watson PH 1996 Immunohistochemical assay for oestrogen receptors in paraffin wax sections of breast carcinoma using a new monoclonal antibody. J Pathol 180:223–227[CrossRef][Medline]
  29. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR 1995 Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182:1545–1556[Abstract/Free Full Text]
  30. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S 1998 A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391:43–50[CrossRef][Medline]
  31. Yamazaki Y, Tsuruga M, Zhou D, Fujita Y, Shang X, Dang Y, Kawasaki K, Oka S 2000 Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis. Exp Cell Res 259:64–78[CrossRef][Medline]
  32. Mashima T, Naito M, Tsuruo T 1999 Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis. Oncogene 18:2423–2430[CrossRef][Medline]
  33. Kokawa K, Shikone T, Nakano R 1996 Apoptosis in the human uterine endometrium during the menstrual cycle. J Clin Endocrinol Metab 81:4144–4147[Abstract/Free Full Text]
  34. Tao XJ, Tilly KI, Maravei DV, Shifren JL, Krajewski S, Reed JC, Tilly JL, Isaacson KB 1997 Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J Clin Endocrinol Metab 82:2738–2746[Abstract/Free Full Text]
  35. Kodama A, Lechler T, Fuchs E 2004 Coordinating cytoskeletal tracks to polarize cellular movements. J Cell Biol 167:203–207[Abstract/Free Full Text]
  36. Gourlay CW, Ayscough KR 2005 The actin cytoskeleton: a key regulator of apoptosis and ageing? Nat Rev Mol Cell Biol 6:583–589[CrossRef][Medline]
  37. Kumazawa Y, Kawamura K, Sato T, Sato N, Konishi Y, Shimizu Y, Fukuda J, Kodoma H, Tanaka T 2005 HCG up-regulates survivin mRNA in human granulosa cells. Mol Hum Reprod 11:161–166[Abstract/Free Full Text]
  38. Kayisli UA, Selam B, Guzeloglu-Kayisli O, Demir R, Arici A 2003 Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system. J Immunol 171:2305–2313[Abstract/Free Full Text]
  39. Castro A, Johnson MC, Anido M, Cortinez A, Gabler F, Vega M 2002 Role of nitric oxide and bcl-2 family genes in the regulation of human endometrial apoptosis. Fertil Steril 78:587–595[CrossRef][Medline]
  40. Tsujimoto Y, Shimizu S 2000 Bcl-2 family: life-or-death switch. FEBS Lett 466:6–10[CrossRef][Medline]
  41. Croy S, Adams JM 2002 The BCL2 family: regulators of cellular life or death switch. Nat Rev Cancer 2:647–656[CrossRef][Medline]
  42. Radtke F, Schweisguth F, Pear W 2005 The Notch ‘gospel.’ EMBO Rep 6:1120–1125[CrossRef][Medline]
  43. Peng X, Kim JJ, Fazleabas AT 2000 Apoptosis and differentiation in baboon stromal cells: a role for chorionic gonadotrophin? Biol Reprod 62(Suppl 1):307–308 (Abstract 510)
  44. Shelly LL, Fuchs C, Miele L 1999 Notch-1 inhibits apoptosis in murine erythroleukemia cells and is necessary for differentiation induced by hybrid polar compounds. J Cell Biochem 73:164–175[CrossRef][Medline]
  45. Jehn BM, Bielke W, Pear WS, Osborne BA 1999 Cutting edge: protective effects of notch-1 on TCR-induced apoptosis. J Immunol 162:635–638[Abstract/Free Full Text]
  46. Lin DX, Lei ZM, Li X, Rao ChV 2005 Targeted disruption of LH receptor gene revealed the importance of uterine LH signaling. Mol Cell Endocrinol 234:105–116[CrossRef][Medline]
  47. Correia-da-Silva G, Bell SC, Pringle JH, Teixeira NA 2005 Patterns of expression of Bax, Bcl-2 and Bcl-xL in the implantation site in rat during pregnancy. Placenta 26:796–806[CrossRef][Medline]
  48. Yu R, Ren SG, Melmed S 2002 Proteasome inhibitors induce apoptosis in growth hormone- and prolactin-secreting rat pituitary tumor cells. J Endocrinol 174:379–386[Abstract]
  49. Akcali KC, Gibori G, Khan SA 2003 The involvement of apoptotic regulators during in vitro decidualization. Eur J Endocrinol 149:69–75[Abstract]
  50. Fazleabas AT, Verhage HG, Waites G, Bell SC 1989 Characterization of an insulin-like growth factor binding protein, analogous to human pregnancy-associated secreted endometrial {alpha}1-globulin, in decidua of the baboon (Papio anubis) placenta. Biol Reprod 40:873–885[Abstract]
  51. Kim JJ, Jaffe RC, Fazleabas AT 1998 Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human. Biol Reprod 59:160–168[Abstract/Free Full Text]
  52. Kessler CA, Moghadam KK, Schroeder JK, Buckley AR, Brar AK, Handwerger S 2005 Cannabinoid receptor I activation markedly inhibits human decidualization. Mol Cell Endocrinol 229:65–74[CrossRef][Medline]
  53. Firth SM, Baxter RC 2002 Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 23:824–854[Abstract/Free Full Text]
  54. Clemmons DR, Maile LA 2005 Interaction between insulin-like growth factor-I receptor and {alpha}Vß3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs. Mol Endocrinol 19:1–11[Medline]
  55. Gleeson LM, Chakraborty C, McKinnon T, Lala PK 2001 Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through {alpha}5ß1 integrin via mitogen-activated protein kinase pathway. J Clin Endocrinol Metab 86:2484–2493[Abstract/Free Full Text]
  56. Szmidt M, Cameo P, Strakova Z, Mavrogianis P, Sharpe-Timms KL, Fazleabas AT 2005 Regulation of the endometrial chorionic gonadotrophin receptor (CGR) during the menstrual cycle and early pregnancy in the baboon. J Soc Gynecol Investig 12(Suppl 2):190A (Abstract 328)



This article has been cited by other articles:


Home page
 EndocrinologyHome page
P. Banerjee, K. Sapru, Z. Strakova, and A. T. Fazleabas
Chorionic Gonadotropin Regulates Prostaglandin E Synthase via a Phosphatidylinositol 3-Kinase-Extracellular Regulatory Kinase Pathway in a Human Endometrial Epithelial Cell Line: Implications for Endometrial Responses for Embryo Implantation
Endocrinology, September 1, 2009; 150(9): 4326 - 4337.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
H. Fluhr, S. Krenzer, G. M. Stein, B. Stork, M. Deperschmidt, D. Wallwiener, S. Wesselborg, M. Zygmunt, and P. Licht
Interferon-{gamma} and tumor necrosis factor-{alpha} sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis
J. Cell Sci., December 1, 2007; 120(23): 4126 - 4133.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
O. Eyal, J.-B. Jomain, C. Kessler, V. Goffin, and S. Handwerger
Autocrine Prolactin Inhibits Human Uterine Decidualization: A Novel Role for Prolactin
Biol Reprod, May 1, 2007; 76(5): 777 - 783.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
P. Cameo, M. Szmidt, Z. Strakova, P. Mavrogianis, K. L. Sharpe-Timms, and A. T. Fazleabas
Decidualization Regulates the Expression of the Endometrial Chorionic Gonadotropin Receptor in the Primate
Biol Reprod, November 1, 2006; 75(5): 681 - 689.
[Abstract] [Full Text] [PDF]

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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jasinska, A.
Right arrow Articles by Fazleabas, A. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jasinska, A.
Right arrow Articles by Fazleabas, A. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals