help button home button Am J Pathol The FASEB Journal
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
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 Google Scholar
Google Scholar
Right arrow Articles by Rolaki, A.
Right arrow Articles by Makrigiannakis, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rolaki, A.
Right arrow Articles by Makrigiannakis, A.
(American Journal of Pathology. 2007;170:1561-1572.)
© 2007 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2007.060926

Luteogenic Hormones Act through a Vascular Endothelial Growth Factor-Dependent Mechanism to Up-Regulate {alpha}5ß1 and {alpha}vß3 Integrins, Promoting the Migration and Survival of Human Luteinized Granulosa Cells

Alexandra Rolaki*, George Coukos{dagger}, Dimitris Loutradis{ddagger}, Horace M. DeLisser§, Christos Coutifaris{dagger} and Antonis Makrigiannakis*

From the Laboratory of Human Reproduction,* Department of Obstetrics and Gynaecology, Medical School, University of Crete, Heraklion, Greece; the Division of Human Reproduction,{dagger} Department of Obstetrics and Gynaecology, and the Division of Pulmonary, Allergy, and Critical Care,§ University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; the Department of Obstetrics and Gynaecology,{ddagger} Medical School, University of Athens, Athens, Greece


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The formation of the corpus luteum (CL) is critical for the establishment of a successful pregnancy. After ovulation, the CL develops from the remnants of the ovulated ovarian follicle. This process, which involves varying cell-matrix interactions, is poorly characterized. To understand the role and potential regulation of cell-matrix interactions in the formation of the CL, we investigated the expression and activity of the matrix protein fibronectin (FN) and several of its integrin receptors on luteinized granulosa cells (GCs). In situ, FN and several FN-binding integrins were detected around luteinizing GCs during the early luteal phase, although expression declined in the late luteal phase. In vitro, GCs released FN, and stimulation of these cells with human chorionic gonadotropin increased the surface expression of FN, {alpha}5ß1, and {alpha}vß3. Up-regulation of these proteins on GCs was reproduced by stimulation with vascular endothelial growth factor (VEGF) and was inhibited by anti-VEGF antibody. Lastly, expression of {alpha}5ß1 and {alpha}vß3 mediated adhesion to FN, facilitated migration, and prevented apoptosis. These data suggest that in vivo luteogenic hormones, in part through a VEGF-dependent mechanism, stimulate selected integrin-matrix adhesive interactions that promote the motility and survival of GCs and thus contribute to the formation and preservation of the CL.

At the onset of the adult female reproductive life, the normal ovary carries several hundreds of thousands of primordial follicles,1 which are formed during embryonic life. At the beginning of every normal menstrual cycle, a cohort of these primordial follicles begins to grow, but only one follicle will complete the maturation cycle and become dominant, developing into a large preovulatory follicle. Late in its maturation, the dominant follicle produces increasing amounts of estradiol, which provides the crucial stimulus for a surge in luteinizing hormone (LH) that completes the maturation of the dominant follicle and triggers the rupture of the follicle and release of the oocyte (ovulation).

After expulsion of the oocyte, structural changes of granulosa and thecal cells are provoked, resulting in transformation of the collapsed follicle into a highly vascularized endocrine gland, the corpus luteum (CL). The main hormone product of the CL is progesterone, which induces the necessary endometrial modifications required for the acquisition of a receptive state, an anticipation of embryo implantation. However, the CL has a short life span, degenerating over the course of 2 weeks into a fibrous hormonally inactive residue. During corpus luteum regression, granulosa and lutein cells undergo apoptosis, a mechanism modulated by many different factors.2,3 If conception and successful implantation of the embryo occur, the corpus luteum is preserved for an additional 8 to 12 weeks. This is a critical condition for the establishment and maintenance of pregnancy because the CL is the main source of vital steroidogenic hormones supporting the gestation. The extended life of the CL is accomplished by the trophoblastic production of the luteotropic hormone human chorionic gonadotropin (hCG)4 through the same receptor as LH.5 Although some of the biochemical and endocrine events characterizing the formation and regression of CL have been well established,6,7 the molecular aspects underlying the migration and survival of luteinized granulosa cells (GCs) and the endocrine/paracrine mechanisms by which LH and hCG act on GCs to transform the ruptured follicle into the CL are not well characterized.

A number of studies have established for many cell types that the binding of cell surface integrins to their ligands in the extracellular matrix facilitates cell migration, proliferation, and survival.8-11 It has been reported that human GCs express the {alpha}5ß1 integrin and its ligand fibronectin (FN),12 but it is unknown whether this or other integrin-matrix interactions have important functional consequences for GCs. It has also been demonstrated that GCs produce vascular endothelial growth factor (VEGF)13-17 in response to stimulation with hCG. This activity was confirmed by us as well (Table 1) . Moreover, VEGF has been reported to increase the expression of integrins in other cell systems.18,19 Expression of VEGF and its receptor (Flt-1) has been previously reported in human lutein cells in functioning corpora lutea.20 Recently, mRNA for VEGF and its receptors Flt-1 and KDR/Flk-1 was detected in human corpora lutea during the luteal phase.21 We therefore investigated the expression of selected FN-binding integrins on luteinizing GCs, the potential regulation of that expression by luteogenic hormone, and the possible involvement of mitogenic VEGF. In addition, we studied the involvement of these receptors in promoting the adhesion, the migration, and survival of GCs.


View this table:
[in this window]
[in a new window]

  		Table 1. VEGF Release from GCs

 

   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents, Antibodies, Ligands, and Chemicals

All reagents were of analytical grade and were purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise stated. The following monoclonal antibodies (mAbs) were obtained from Chemicon International Inc. (Temecula, CA): blocking mAbs to the integrin {alpha}5ß1 (JBS5), {alpha}vß3 (LM 609), and {alpha}vß5 (PIF6) receptors; anti-{alpha}5ß1-integrin-activating mAb (HA5); anti-FN; anti-platelet endothelial cell adhesion molecule-1 (P2B1); and anti-human leukocyte antigen-DR. mAb to Flt-1 (VEGF receptor-1 [VEGFR-1]) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-human VEGF-neutralizing antibody and a second anti-Flt-1 antibody were obtained from R&D Systems Inc. (Minneapolis, MN). Goat anti-serum to mouse whole IgG and nonimmune murine IgG1 were purchased from Cappel Research Products (Durham, NC). Antibodies were used at a concentration of 10 µg/ml purified IgG. Human laminin-1 was purchased from GIBCO BRL Life Technologies (Grand Island, NY). Purified human FN, {alpha}5ß1, {alpha}vß3, and {alpha}vß5 proteins were purchased from Chemicon International Inc. Purified Flt-1 peptide antigen was obtained from Santa Cruz Biotechnology.

Cell Culture and Treatments

Human GCs were isolated from 16 patients, aged 25 to 43 years old, undergoing in vitro fertilization/embryo transfer. These cells had been exposed in vivo to a follicular recruitment regimen including a gonadotropin-releasing hormone agonist (Lupron) for pituitary suppression and purified follicle-stimulating hormone (Fertinex; Serono, Randolph, MA) for follicular stimulation. Moreover, all patients had received a single dose of purified hCG (10.000 IU) 36 hours before follicular aspiration. The present study was approved by the Hospital of University of Pennsylvania, and informed consent was obtained from all patients before tissue collection.

The follicular fluid was collected and centrifuged. The sedimented cells were resuspended in calcium- and magnesium-free Hanks’ balanced salt solution (Gibco BRL), overlayed on Ficoll-Paque (Pharmacia Biotechnology, Uppsala, Sweden), and centrifuged at 400 x g for 30 minutes. The cells were collected from the interphase and cultured in Hams F-12/Dulbecco’s modified Eagle’s medium [1:1 (v/v); Gibco BRL] media supplemented with 10% fetal bovine serum, penicillin (10 IU/ml), streptomycin (0.05 mg/ml), and Fungizone (0.25 mg/ml), as previously described.22 In every culture dish, 2 x 105 cells were plated, which were treated with the usual pharmacological dose of VEGF (100 ng/ml) and with urinary derived hCG (1 IU/ml) without and with anti-human VEGF-neutralizing antibody (6 µg/ml), in serum-free Hams F12/Dulbecco’s modified Eagle’s medium [1:1 (v/v)] medium with 0.2% bovine serum albumin (BSA) and 10 mmol/L HEPES. The cells were cultured for 48 to 72 hours in media containing hCG to develop an hCG dose response before being treated with antibodies. The ratio of contaminating monocytes, identified by the anti-CD14 mAb (Becton Dickinson, Lincoln Park, NJ) was <1.6%. Each experiment was performed on at least three separate occasions with different cell preparations to ensure consistency of the findings.

Immunohistochemistry

Paraffin-embedded human ovarian tissue sections were deparaffinized at 70°C in xylene, rehydrated, and rinsed in phosphate-buffered saline (PBS) (pH 7.4). Sections were treated with 0.1% hydrogen peroxide for 30 minutes at 20°C, washed in PBS, and digested with prewarmed pepsin (0.65 mg/ml in PBS) at 40°C for 5 minutes. They were preincubated with 5% blocking serum (normal goat serum; Vector Laboratories, Burlingame, CA) and then incubated with the primary antibody (10 µg/ml) for 1 hour at room temperature. Localization of the primary antibody was performed by incubation of the sections with a biotinylated anti-mouse IgG antibody, and then biotin was detected using an avidin-biotin-peroxidase kit (Vector Laboratories) with diaminobenzidine as the chromogenic substrate. Negative control sections were processed in an identical manner by substitution of the primary antibody with a purified mouse IgG fraction and preabsorption of the primary antibody with the target antigen to assess the specificity of the primary antibody. Various samples of archival ovarian tissue specimens at various phases of the follicular cycle were selected from patients who had undergone oophorectomy as part of pelvic operations for benign gynecological disease. Dating was based on endometrial tissue specimens from the same patients, according to Noyes criteria.23

The intensity and distribution patterns of the staining reaction were evaluated by two blinded, independent observers using the semiquantitative immunoreactive score, as previously described.24 The immunoreactive score was calculated by multiplication of optical staining intensity (graded as 0, no; 1, weak; 2, moderate; and 3, strong staining) and the percentage of positive stained cells (0, no staining; 1, <10% of the cells; 2, 11 to 50% of the cells; 3, 51 to 80% of the cells; and 4, >81% of the cells).

Flow Cytometric Analysis of Human Luteinizing GCs

Flow cytometry was performed as described elsewhere.22 The isolated human GCs were washed in 0.1% BSA/PBS, centrifuged, and incubated with the monoclonal anti-FN antibody and the mAbs anti-{alpha}5ß1 (HA5), anti-{alpha}vß3 (LM609), and anti-{alpha}vß5 (PIF6); the purified mouse IgG3 (control, 100 µg/ml) for 1 hour at 4°C; or the integrin peptide for 30 minutes in room temperature. The cells were then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG, for 30 minutes at 4°C in the dark, rinsed in PBS, and analyzed using an EPICS XL flow cytometer (Coulter Corporation, Hialeah, FL). The ratio of contaminating monocytes, identified by the anti-CD14 mAb (Becton Dickinson), was <1.6%.

Immunofluorescence

Sterile glass coverslips were coated with extracellular matrix ligands as previously described, with minor modifications.23 In brief, the coverslips were incubated overnight with FN (50 µg/ml), vitronectin (20 µg/ml), or laminin (20 µg/ml) in PBS at 4°C and washed with PBS, and the GCs were plated onto them. Cells grown on coverslips were washed twice in prewarmed (37°C) Dulbecco’s modified Eagle’s medium and twice in prewarmed PBS containing 1.5 mmol/L Ca2+ and fixed in 100% methanol (–20°C for 5 minutes). Cells were incubated in 10% normal goat serum (30 minutes at room temperature) and then with the primary antibodies (2 hours at room temperature) at 10 µg/ml in 10% goat serum. A fluorescein-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA) was used at 1:200 (30 minutes at room temperature). Negative control sections were processed in an identical manner by substitution of the primary antibody with PBS. Coverslips were mounted on glass slides with Fluormount G (Fisher Scientific, Malvern, PA) contain-ing 1,4-diazabicyclo[2.2.2]octane (Polysciences Inc., Warrington, PA) to stabilize fluorescence and photographed with a Nikon microscope (Tokyo, Japan).

Adhesion Assays

Tissue culture plastic dishes (100 and 60 mm; Corning Co., Corning, NY) or sterile glass coverslips were coated with extracellular matrix ligands as previously described, with minor modifications.23 In brief, tissue culture plastic dishes or coverslips were incubated with 50 µg/ml FN in PBS or 100 µg/ml poly-L-lysine in PBS for 16 hours at 4°C. Plates were then washed three times with PBS and blocked with 1% heat-denatured BSA/PBS for 1 hour at room temperature. GCs were starved with methionine-free media for 2 hours and subsequently incubated with [35S]methionine-containing media overnight. GCs were subsequently harvested by short exposure to trypsin, washed with serum-free media containing soybean trypsin inhibitor, centrifuged, and resuspended in media containing 1% fetal bovine serum. Cells (5 x 103) were seeded on the different coated plates and allowed to interact with them for 30 minutes at 37°C. Cells were then washed in PBS and lysed with Tris-HCl media containing 1% sodium dodecyl sulfate. Lysates were then transferred in scintillation vials, and radioactivity was counted. Estimation of adhesion was performed in a similar manner by calculating the amount of radioactivity in each well.

Migration Assays

Time-lapse video microscopy was performed as previously described.22 Human GCs were cultured in serum-free medium. For these studies, slides obtained from Lab-Tek (Micro Video Instruments, Arrow, MA), which were sealed with a mixture of petroleum jelly and paraffin (20:1) to maintain pH of the medium, were coated with FN (10 µg/ml) for 1 hour at 37°C and blocked with 2% BSA for 1 hour at 37°C. The cells were plated on the slides at a density of 0.5 x 105 cells and then placed in a 37°C-humidified Plexiglas microscope culture chamber (Nikon). A field containing several GCs was selected and observed under phase contrast for 7 hours. Motile activity was studied by measuring the total distance covered by the migrating cells using time-lapse video microscopy. Sequential images were collected at 1-hour intervals. A minimum of 70 cells were studied in three separate experiments for each condition.

Cell Adhesion to Immobilized Anti-Integrin Antibodies

To evaluate the effects of individual integrins, plastic dishes were first coated with 50 µg/ml goat anti-mouse IgG in PBS, at 37°C for 2 hours, rinsed with PBS, blocked with 1% heat-denatured BSA/PBS at 37°C for 1 hour, and then incubated overnight at 4°C with mouse monoclonal antibodies raised against {alpha}5ß1, {alpha}vß3 integrin, or HLA-DR at a concentration of 40 µg/ml in PBS. GCs were harvested with ethylenediamine tetraacetic acid, washed, and resuspended in adhesion buffer (20 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 2 mmol/L MgCl2, and 5 mmol/L sodium pyruvate, pH 7.4). Cells were then allowed to attach to antibody-coated dishes at 37°C.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick-End Labeling Assay

Apoptosis was detected by in situ 3'-end labeling of DNA fragments in vitro. DNA fragments were labeled and detected by use of the reagents and procedures provided in the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). In brief, GCs grown on ligand-coated coverslips in serum-free medium for various time points were fixed in 10% buffered formalin and washed twice in PBS. The cells were then incubated in a humidified chamber at 37°C for 1 hour in the presence of terminal deoxynucleotidyl transferase and digoxigenin-11 dUTP and dATP, washed in buffer, and incubated with anti-digoxigenin-fluorescein antibody for 30 minutes at room temperature. The cells were then washed in buffer and observed under epifluorescence and bright-field optics. The nuclear structures of individual cells were stained with propidium iodide. The number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive nuclei was counted in an average of 12 randomly selected high-power fields (x40), and the results were expressed as percentage of positive cells. An average of 250 cells was counted for each coverslip. If size and position of positively stained nuclear fragments suggested that they were remnants of a single cell, they were recorded as one cell.

Flow Cytometric Analysis of DNA Content

Detection of apoptosis by flow cytometric analysis of DNA content was performed as previously described.22 Trypsin-generated GC suspensions (including floating cells) or cells from suspension cultures (minimum 106) were fixed in 70% ethanol for at least 16 hours, treated with RNase A (500 µg/ml for 30 minutes at room temperature; Sigma), stained with propidium iodide (PI 20 µg/ml), and analyzed using an EPICS XL flow cytometer (Coulter Corporation). Data were analyzed using a Cellfit program (Wistar Institute, Philadelphia, PA). Cells that contained less than 2n DNA content in the cell cycle analysis profile were considered to be apoptotic.

Determination of VEGF Concentration

The concentration of VEGF in the media of cultured GCs was determined by enzyme-linked immunosorbent assay using a commercially available kit (R&D Systems, Minneapolis, MN).

Statistical Analysis

In TUNEL assays, statistical comparisons between the different treated groups were performed using Student’s t-test. In adhesion and migration experiments, the differences among the mean values for different groups were evaluated by one-way analysis of variance followed by Student’s t-test. In the flow cytometric analysis, the differences of positivity rate and mean fluorescence intensity of integrins {alpha}5ß1, {alpha}vß3, and {alpha}vß5 and fibronectin expression on GCs were analyzed by the Mann-Whitney test. Data are expressed as mean ± SE, and P values <0.05 were considered to be significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of FN and Several of Its Integrin Receptors on GCs during the Menstrual Cycle

To determine the expression of FN and three of its integrin receptors, {alpha}5ß1, {alpha}vß3, and {alpha}vß5, during the CL formation, immunohistochemical staining was performed on ovarian tissue from different phases of luteogenesis (Figure 1) . For these studies, well-characterized antibodies were used (see Materials and Methods), and the binding of each was determined to be specifically inhibited by preabsorption with the target antigen. No staining was detected in small preantral follicles, whereas there was a clear association of all these molecules with CL formation. In the CL of the early luteal phase (menstrual cycle day 15 to 18, n = 14), FN was detected in the matrix, and {alpha}5ß1, {alpha}vß3, and, to a much lesser extent, {alpha}vß5 were expressed on luteinizing GCs, expression that was reduced or absent in the late luteal phase (days 24 to 28, n = 12). These data suggest that CL formation is associated with deposition of FN in the matrix surrounding GCs along with expression of FN binding integrins by these cells.


Figure 1
View larger version (109K):
[in this window]
[in a new window]

  		Figure 1. In situ staining of ovarian tissue for expression of fibronectin and several of its integrin receptors on GCs. Ovarian tissue was stained with antibodies against FN (A–C), {alpha}5ß1 (D–F), {alpha}vß3 (G–I), and {alpha}vß5 (J–L) to determine the expression of these molecules in developing follicles and during corpus luteum formation and regression. In developing follicles (A, D, G, and J; arrows), fibronectin, {alpha}5ß1, {alpha}vß3, and {alpha}vß5 were not detected on the GCs. In contrast, there was obvious staining for FN, {alpha}5ß1, and {alpha}vß3 on the GCs of early luteal phase (B, E, and H), expression that was reduced or absent during the late luteal phase (C, F, and I). For {alpha}vß5, marginal staining was noted during the early luteal phase (K), but none was evident later (L). Scale bars = 100 µm. M: Staining intensity was determined by the semiquantitative immunohistochemical immunoreactive score (IRS). Data represent mean ± SEM.

 
Expression by GCs of FN and Its Receptors {alpha}5ß1, {alpha}vß3, and {alpha}vß5 Integrins in Vitro

To confirm the immunohistochemical data, expression of FN and {alpha}5ß1, {alpha}vß3, and {alpha}vß5 integrins on freshly isolated luteinizing GCs was assessed by flow cytometry (Figure 2) . These cells have been previously exposed to high doses of circulating hCG in vivo (see Materials and Methods) and display features of luteinized granulosa cells (GCs).1 There was significant expression of the {alpha}5ß1 and {alpha}vß3 integrins on freshly isolated GCs (detected on 61 ± 10% and 38 ± 5% of cells, respectively; n = 16), whereas a much lower percentage of cells (8.8 ± 1%; n = 16) expressed {alpha}vß5 integrin. FN was also detected on the surface of GCs (expressed on 52% ±10 of cells; n = 16), suggesting that some of the integrin receptors were occupied by FN.


Figure 2
View larger version (99K):
[in this window]
[in a new window]

  		Figure 2. GC surface expression of FN and three fibronectin-binding integrin receptors, {alpha}5ß1, {alpha}vß3, and {alpha}vß5. Immunofluorescence staining of GCs cultured on vitronectin (A) or fibronectin (B–E) under serum-free conditions and stained for FN (A), {alpha}5ß1 (B), {alpha}vß3 (C), and {alpha}vß5 (D) is shown. E: Negative control is also shown (without the primary antibody). For GCs plated on vitronectin, FN was detected on the surface of these cells on their filopodia (arrows). {alpha}5ß1, {alpha}vß3, and {alpha}vß5 integrins were also expressed on the surface of GCs plated on FN, but only {alpha}5ß1 and {alpha}vß3 formed focal adhesions (arrowheads). Scale bars = 20 µm. F: The surface expression of FN, {alpha}5ß1, {alpha}vß3, and {alpha}vß5 on human GCs was also determined by flow cytometry. Filled and unfilled tracings represent, respectively, the background staining and staining for the targeted protein. Fibronectin was also detected on the surface of GCs and along with significant expression of the {alpha}5ß1 and {alpha}vß3 integrins. A small percentage cells expressed {alpha}vß5. These data are representative of more than 10 independent experiments.

 
Surface expression of FN and its receptors {alpha}5ß1, {alpha}vß3, and {alpha}vß5 was further assessed by immunofluorescence staining of GCs cultured on various matrix proteins under serum-free conditions (Figure 2) . FN was detected on the surface of GCs plated on vitronectin or laminin, but it was localized mainly on the projections of the cells. Because these cells were cultured in the absence of serum, these data suggest that GCs secrete FN, some of which becomes associated with the cell surface. {alpha}5ß1, and {alpha}vß3 integrins were also expressed on the surface of GCs, whereas a lower percentage of cells expressed {alpha}vß5 (a number of cells expressing the protein is shown). As has been previously reported in other cell types,25,26 focal adhesion formation was noted with {alpha}5ß1 and {alpha}vß3 but not {alpha}vß5.

hCG Increases the Surface Expression of FN and Its Receptors {alpha}5ß1 and {alpha}vß3 on GCs

We next investigated the effect of hCG on the expression of FN and its integrin receptors on luteinizing GCs by culturing these cells in the absence or presence of hCG for 48 to 72 hours. As assessed by flow cytometry, levels of surface expression of these proteins in unstimulated cells did not differ significantly from that seen on freshly isolated cells. In contrast, stimulation with hCG led to a significant increase in the number of cells expressing FN compared with untreated controls (88 ± 3.6 versus 52 ± 2.7% of cells, P < 0.05), {alpha}5ß1 compared with control (90 ± 2.5 versus 60 ± 1.9%, P < 0.05), and {alpha}vß3 compared with control (78 ± 1.0 versus 38 ± 3.2%, P < 0.05) but had no effect on {alpha}vß5 expression compared with control (8.8 ± 1.8 versus 8.0 ± 1.7%). In addition, exposure to hCG was followed by an up-regulation of FN, {alpha}5ß1, and {alpha}vß3 as indicated by a two- to fourfold increase in the mean fluorescence intensity.

VEGF Increases the Expression of FN, {alpha}5ß1, and {alpha}vß3 on Cultured GCs

We next investigated the effect of VEGF on the expression of FN and its integrin receptors on GCs by culturing these cells in the absence or presence of VEGF165. As determined by flow cytometry, stimulation with VEGF165 increased the number of cells expressing FN on the cell surface compared with control (75 ± 2.2 versus 50 ± 2%, P < 0.05), {alpha}5ß1 compared with control (74 ± 3.2 versus 55 ± 2.4%, P < 0.05), and {alpha}vß3 compared with control (68 ± 2 versus 38 ± 2.6%, P < 0.05) without changes in {alpha}vß5 (8.8 ± 1 versus 8.7 ± 1%, P < 0.05). In addition, FN, {alpha}5ß1, and {alpha}vß3 expression was also increased by approximately two- to fourfold as indicated by an increase in the mean fluorescence intensity.

Anti-VEGF Antibody Inhibits the Increase in Expression of FN, {alpha}5ß1, and {alpha}vß3 Induced by hCG

Given our findings that both hCG and VEGF increase the expression of FN and FN-binding integrins on the surface of GCs, we investigated whether the effects of hCG might be mediated by VEGF (Figure 3) . GCs stimulated with hCG were therefore cultured in the absence or presence of neutralizing antibody against VEGF. This antibody, which is able to neutralize more than 90% of the VEGF released in response to hCG stimulation (Table 1) , almost completely inhibited the hCG-induced up-regulation of FN, {alpha}5ß1, and {alpha}vß3 on GCs (Figure 3) . After addition of VEGF antibody (100 ng/ml), the effects of anti-VEGF were inhibited (data not shown). Together these data suggest that hCG increases the expression of these integrin receptors, at least in part, in an autocrine/paracrine manner through the secretion of VEGF from GCs and its subsequent binding to VEGFR on GCs. Supporting this proposal are the previous reports21 and our findings that GCs express the VEGF receptor (Flt-1) in vitro and in situ (data not shown).


Figure 3
View larger version (30K):
[in this window]
[in a new window]

  		Figure 3. Effect of anti-VEGF antibody on the changes in expression of fibronectin, {alpha}5ß1, and {alpha}vß3 induced by hCG. Flow cytometry was used to assess the expression of fibronectin (A, D, and G), {alpha}5ß1 (B, E, and H), and {alpha}vß3 (C, F, and I) on unstimulated, control GCs (A–C) and GCs stimulated with hCG in the absence (D–F) or presence of antibody against VEGF (G–I). The dotted line indicates the peak fluorescence intensity of the control cells. Using this as a reference, hCG increased the expression of all three proteins (P < 0.05, compared with control), an effect that was inhibited by inclusion of anti-VEGF antibody. Control antibody that bound to GCs did not have any effect (data not shown). These data are representative of three independent experiments.

 
Adhesion of GCs to FN Is Inhibited by Antibody to {alpha}5ß1 and {alpha}vß3

To access the functional significance of {alpha}5ß1 and {alpha}vß3 expression on GCs, we studied the adhesion of these cells to plastic surfaces coated with purified FN or with poly-L-lysine (PLL) for an integrin-independent cell adhesion. It was observed that the adhesion of GCs to FN was significantly greater than adhesion to uncoated or PLL-coated surfaces and was inhibited by antibodies against {alpha}5ß1 and {alpha}vß3 (Figure 4) . Antibody against {alpha}vß5 was not inhibitory. After stimulation with hCG, there was 1.5- to 2.0-fold increase in the adhesion of GCs to FN with a comparable pattern of antibody inhibition.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		Figure 4. Adhesion of GCs to fibronectin. The adhesion of unstimulated GCs and GCs stimulated with hCG to uncoated plastic surfaces or plastic surfaces coated with PLL or FN was studied. For unstimulated GCs, there was significantly more adhesion to the FN surface than to the uncoated or PLL surfaces (*P < 0.05), adhesion that was significantly increased following stimulation with hCG (**P < 0.05). The adhesion mediated by both unstimulated and stimulated cells was inhibited by the inclusion in the media of antibody against {alpha}5ß1 and/or {alpha}vß3 (***P < 0.05) but not against {alpha}vß5.

 
hCG Stimulates GCs Migration on FN That Is Inhibited by Antibody to {alpha}5ß1 and {alpha}vß3

Given the finding that both {alpha}5ß1 and {alpha}vß3 mediate adhesion of GCs to FN, we investigated whether these integrins were also involved in the migration of GCs on FN, phenomena very much dependent on integrin-matrix adhesion. Time-lapse video microscopy was used to follow the random motile activity of GCs plated on FN. We observed that stimulation with hCG increased the migratory behavior of GCs, consistent with the hCG-induced increase in integrin expression, and that compared with control, antibody against {alpha}5ß1 and {alpha}vß3 either singly or combined significantly inhibited the migratory activity of these cells (Figure 5) .


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		Figure 5. Migration of GCs on fibronectin. The migration of unstimulated GCs and GCs stimulated with hCG on plastic surfaces coated with fibronectin was studied. Stimulation of GCs in the absence of antibody (control) with hCG significantly increased the migratory activity of GCs compared with unstimulated cells (*P < 0.05). The movements of both unstimulated and stimulated GCs were inhibited by antibody against {alpha}5ß1 and/or {alpha}vß3 (**P < 0.05) but not by mouse IgG.

 
{alpha}5ß1 and {alpha}vß3 Integrin-Dependent Adhesion of GCs Inhibits Apoptosis

Integrin-mediated attachment to matrix has been shown to protect cells against the process of programmed cell death or apoptosis.27,28 We therefore investigated the activity of {alpha}5ß1 and {alpha}vß3 integrins in promoting the survival of GCs cultured in the absence of serum, conditions known to induce apoptosis22,29 (Figure 6 ; Table 2 ). Cell survival was studied either by in situ detection of DNA fragmentation using the TUNEL assay or flow cytometric analysis of DNA content. GCs cultured in the absence of a substratum or on PLL underwent significant apoptosis after 24 hours under serum-free conditions (Figure 6) . In contrast, GCs grown on FN without serum showed significantly lower rates of apoptosis at 24 hours (P < 0.05 versus PLL). Co-incubation with blocking antibodies (included in the media) against {alpha}5ß1 and/or {alpha}vß3 increased apoptosis (P < 0.05). Very similar results were obtained by flow cytometric analysis of DNA content (Table 2) . In additional experiments, using activated anti-{alpha}5ß1 and/or anti-{alpha}vß3 antibodies to mimic specific ligand binding, the levels of apoptosis were comparable with that seen by GCs plated on FN. Together these data suggest that in vivo, the adhesion of GCs to FN through the {alpha}5ß1 and {alpha}vß3 integrins prevents apoptosis and thus may promote their survival (Figure 7) .


Figure 6
View larger version (97K):
[in this window]
[in a new window]

  		Figure 6. Apoptosis of GCs cultured on fibronectin in serum-free conditions. GCs were plated on FN in the absence of serum, and the level of apoptosis was assessed by TUNEL assay. Shown are the phase contrast images (A, C, and E), the corresponding TUNEL staining (B, D, and F), and the quantitation of the level of apoptosis (G) as expressed by the percentage of TUNEL assay-positive cells [% TUNEL (+) GCs]. GCs underwent significant apoptosis after 24 hours of culture under serum-free conditions on PLL (C, D, and G) as evidenced by the presence of cells with TUNEL-positive nuclei (arrows). The extent of apoptosis was significantly less when the GCs were grown on FN (A, B, and G) (*P < 0.05 compared with PLL) with a loss of the survival effects on FN if antibody against {alpha}5ß1 (E–G) and/or {alpha}vß3 (G) was included in the media (**P < 0.05 compared with FN alone). Scale bars = 20 µm.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Apoptosis of GCs Cultured on Fibronectin in Serum-Free Conditions Assessed by Flow Cytometric Analysis of DNA Content

 

Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		Figure 7. Apoptosis of the GCs cultured on immobilized anti-integrin antibody in serum-free conditions. GCs were plated on plastic surfaces bearing activated anti-{alpha}5ß1 or -{alpha}vß3 antibodies in the absence of serum, and the level of apoptosis was assessed by determining the level of apoptosis as expressed by the percentage of TUNEL assay-positive cells [% TUNEL (+) GCs]. Under these conditions, the level of apoptosis after attachment to {alpha}5ß1 and {alpha}vß3 antibodies was comparable with that observed after attachment to FN and was significantly different from that seen with control antibody to HLA-DR (*P < 0.05 compared with anti-HLA-DR).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Increasing evidence indicates the contribution of cell-cell adhesion to the maturation and integrity of the human CL.22,30 However, the interactions of luteinized granulosa cells with the extracellular matrix during CL formation are not well understood. Therefore, the expression and activity of the matrix protein FN and several of its integrin receptors on human GCs were investigated. In situ, FN was detected around GCs, whereas several FN-binding integrins along with the VEGFR, Flt-1, were expressed on the surface of these cells in association with formation of the CL. In vitro, GCs released FN, and stimulation of these cells with the luteogenic hormone hCG or VEGF increased the surface expression of {alpha}5ß1 and {alpha}vß3 and the amount of FN associated with the cell surface. Furthermore, the hCG-induced up-regulation of FN, {alpha}5ß1, and {alpha}vß3 on the surface of GCs was inhibited by anti-VEGF antibody. Lastly, expression of {alpha}5ß1 and {alpha}vß3 mediated adhesion to FN that promoted the migration and survival of GCs.

A critical prerequisite for the establishment of pregnancy is the formation of the CL remnants of the ruptured follicle after release of the oocyte. Consistent with this are the observations from immunostaining of ovarian tissue that FN, a matrix protein involved in many of the morphological and cytological changes of development and tissue repair,31 accumulates within the granulosa cell layer during the early luteal phase (Figure 1) . GCs may be a source of some of the FN found in the CL as cultured GCs were found in vitro to secrete matrix containing FN (Figure 2) .

The best-defined membrane receptor mediators of cellular interactions with the extracellular matrix are the integrins, a family of adhesion molecules.10 Several integrins, such as {alpha}5ß1 and {alpha}vß3, have been reported to mediate adhesion to FN.32 In a pattern that temporally mirrored the accumulation of FN, {alpha}5ß1 and {alpha}vß3 were expressed on GCs during the initial stages of CL formation but not during the late luteal phase preceding regression of the organ (Figure 1) . Our data extend the observations by Honda et al,12 who in a more limited study reported a similar pattern of expression for FN and the {alpha}5 subunit during the menstrual cycle. Furthermore, these data suggest that the surge in LH at the time of ovulation results in the accumulation of FN in the matrix with concomitant up-regulated surface expression of FN-binding integrins on GCs. Supporting this suggestion are our in vitro data that stimulation of cultured GCs with hCG increases their expression of {alpha}5ß1 and {alpha}vß3 but not {alpha}vß5.

VEGF is a potent migration-stimulating mitogenic factor of endothelial cells in vitro and has been convincingly established as one of the principal mediators in vivo of vasculogenesis and angiogenesis.33,34 Human VEGF may exist in one of five different isoforms of 121, 145, 165, 189, and 206 amino acids, with VEGF121 and VEGF165 being the most abundant variants.35 It has been previously reported that suppression of VEGF in the developing follicle is associated with inhibition of follicular angiogenesis and antral follicular development, which results in the inhibition of ovulation.36 Furthermore, recent studies have shown that suppression of VEGF at the early and mid-luteal phase in primates caused no marked morphological change in steroidogenic cells, but the secretion of the main hormonal product of these cells was significantly reduced, and the concentration of follicle-stimulating hormone and LH in the serum was increased.37,38

Our findings that 1) VEGF165 or hCG stimulation of GCs triggers the release of FN and increases {alpha}5ß1 and {alpha}vß3 integrin surface expression (Figure 2) , and 2) anti-VEGF antibody prevented the up-regulation in expression of these molecules after stimulation with hCG (Table 1) suggest that some of the effects of hCG on GCs may be mediated in an autocrine/paracrine manner through hCG-induced release of VEGF and its subsequent binding to the Flt-1 receptor on GCs, whereas the integrity and morphology of these cells are normally maintained. Supporting this proposal are the previous reports that hCG regulates expression of VEGF in GCs13-17 and that Flt-1 is expressed in granulosa lutein cells and endothelial cells in human corpora lutea.21 We also confirmed GC surface expression of Flt-1 receptor using in situ staining of ovarian tissue (data not shown). We would note that our data do not exclude the possibility that some of the effects of hCG on FN secretion and integrin expression may occur independently of VEGF and/or that VEGF derived from other cell types may modulate the behavior of GCs.

The induced expression of {alpha}5ß1 and {alpha}vß3 on GCs is functionally significant. In vitro, the adhesion of luteinized GCs to FN was mediated by {alpha}5ß1 and {alpha}vß3, with the GCs having the capacity to use either integrin to adhere to a FN substrate (Figure 4) . Even by using both antibodies anti-{alpha}5ß1 and anti-{alpha}vß3, the inhibition of the adhesion to FN was not complete (Figure 4) . Therefore, it is suggested that part of the effects of hCG on GC adhesion to FN is mediated through other factors that need to be further investigated. These observations are consistent with the many previous reports that have established the importance of integrins in cell matrix adhesion.39,40 For a variety of cell types, this integrin-mediated adhesion and the subsequent intracellular signaling cascades that are triggered are intimately involved with cell processes such as locomotion, division, and survival.

Cell motility involves a continuous cyclical process9,41 that begins with the cell, at its leading edge, extending membrane processes, lamellipodia (broad, flat protrusions) or filopodia (thin needle-like projections), with subsequent attachment to the substratum through integrin receptors. Dissolution of cell-matrix contacts at the rear of the cell with subsequent detachment completes the process. Integrin-dependent cell-matrix adhesive interactions thus play a central role in cell migration as they couple interactions with the substratum to cytoskeletal elements within the cell.40,42 Our observation that FN concentrates along filopodia of GCs (Figure 2) and our finding that the motility of GCs on FN is inhibited by antibodies against {alpha}5ß1 and {alpha}vß3 (Figure 5) suggest that in the context of an FN-rich matrix, GCs in vivo are able to use these integrins to form adhesive contacts that promote cell movement. However, the migrating activity of GCs on FN was not completely inhibited by anti-{alpha}5ß1 and anti-{alpha}vß3 antibodies (Figure 5) ; thus it is assumed that, at least in part, the effects of hCG on GCs movements may occur independently of integrins, presumably through different adhesion molecules that are expressed on the surface of these cells. These suggestions are consistent with the recent study, which provides evidence that {alpha}5 integrin regulates trophoblast migration through binding with FN of the extracellular matrix.43

In addition to its role in cell migration, it has long been recognized that integrin-mediated matrix attachment is also critically important for their survival.11,44 This has been demonstrated in studies that have shown that epithelial or endothelial cells undergo apoptosis when detached from substrate.27,28 Apoptosis after loss of cell anchorage (anoikis) is related to development and tissue homeostasis. Integrins enhance cell preservation through their interaction with the extracellular matrix.45 The exact mechanisms mediating integrin-induced inhibition of apoptosis are largely unknown. Indirect evidence, however, suggests the existence of postreceptor pathways activated after integrin occupancy that up-regulate survival factors (eg, Bcl-2)28,46 and/or inhibit apoptosis-promoting molecules (eg, Bax or interleukin-1ß converting enzyme).47

The CL is a very short-lived endocrine organ. In the absence of an implanted embryo releasing hCG, the CL undergoes rapid regression. There is compelling evidence that this process is mediated by apoptosis of the luteal cells.22,29,48,49 Prevention of GCs and thecal cell apoptosis is thus highly likely to be fundamental to the preservation of the corpus luteum and the establishment of a viable pregnancy. In this report, we show that engagement of {alpha}5ß1 and {alpha}vß3 integrins through luteinized granulosa cell binding to FN or activated anti-integrin antibody, which mimic specific ligand binding, inhibits apoptosis induced by serum starvation (Figures 6 and 7) . In vitro, hCG prevents apoptosis of cultured rat preovulatory follicles50 and rabbit CL.51 Thus in vivo, after blastocyst implantation, trophoblast-derived hCG may enhance integrin-dependent interactions between FN and GCs that maintain the corpus luteum during early gestation.52

In conclusion the following model is proposed. The binding of luteogenic hormone (LH or hCG) to GCs triggers their release of VEGF and induces the surface expression of VEGFR on these cells. The released VEGF (and possibly VEGF from other sources) in turn binds to the newly expressed VEGFR on the GCs, stimulating the secretion of FN into the surrounding matrix and up-regulating the surface expression of at least two FN-binding integrins, {alpha}5ß1 and {alpha}vß3. Subsequent interactions between FN and these integrins trigger adhesive events and intracellular signaling cascades involved in promoting the migration and survival of GCs, activities that contribute ultimately to the formation and/or persistence of the corpus luteum.


   Footnotes
 
Address reprint requests to Antonis Makrigiannakis, M.D., Ph.D., Laboratory of Human Reproduction, Department of Obstetrics and Gynaecology, Medical School, University of Crete, Heraklion 71110, Greece. E-mail: makrigia{at}med.uoc.gr

Supported by the National Institutes of Health (grants HD-31903 to C.C. and HL-03382 to H.M.D.) and by the Alexander Onassis Foundation (to A.M.).

Accepted for publication January 30, 2007.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grazul-Bilska AT, Redmer DA, Reynolds LP: Cellular interactions in the corpus luteum. Seminar Reprod Endocrinol 1997, 15:383-393
  2. Makrigiannakis A, Coukos G, Christofidou-Solomidou M, Montas S, Coutifaris C: Progesterone is an autocrine/paracrine regulator of human granulosa cell survival in vitro. Ann NY Acad Sci 2000, 900:16-25[Abstract/Free Full Text]
  3. Rolaki A, Drakakis P, Millingos S, Loutradis D, Makrigiannakis A: Novel trends in follicular development, atresia and corpus luteum regression: a role for apoptosis. Reprod Biomed Online 2005, 11:93-103[Medline]
  4. Neill JD, Johansson ED, Knobil E: Patterns of circulating progesterone concentrations during the fertile menstrus cycle and the remainder of gestation in the rhesus monkey. Endocrinology 1969, 84:45-48[Medline]
  5. Funaro A, Sapino A, Ferranti B, Horenstein AL, Castellano I, Bagni B, Garotta G, Malavasi F: Functional, structural and distribution analysis of the chorionic gonadotropin receptor using murine monoclonal antibodies. J Clin Endocrinol Metab 2003, 88:5537-5546[Abstract/Free Full Text]
  6. Makrigiannakis A, Amin K, Coukos G, Tilly LJ, Coutifaris C: Regulated expression and potential roles of p53 and Wilms’ tumor suppressor gene (WT1) during follicular development in the human ovary. J Clin Endocrinol Metab 2000, 85:449-459[Abstract/Free Full Text]
  7. Walz A, Keck C, Weber H: Kissel C, Pietrowski D: Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system. Mol Reprod Dev 2005, 72:98-104[CrossRef][Medline]
  8. Ruoslahti E, Reed JC: Anchorage dependence, integrins, and apoptosis. Cell 1994, 77:477-478[CrossRef][Medline]
  9. Huttenlocher A, Sandborg RR, Horwitz AF: Adhesion in cell migration. Curr Opin Cell Biol 1995, 7:697-706[CrossRef][Medline]
  10. Clark EA, Brugge JS: Integrins and signal transduction pathways: the road taken. Science 1995, 268:233-239[Abstract/Free Full Text]
  11. Frisch SM, Ruoslahti E: Integrins and anoikis. Curr Opin Cell Biol 1997, 9:701-706[CrossRef][Medline]
  12. Honda T, Fujiwara H, Yamada S, Fujita K, Nakamura K, Nakayama T, Higuchi T, Ueda M, Maeda M, Mori T: Integrin {alpha}5 is expressed on human luteinizing granulosa cells during corpus luteum formation, and its expression is enhanced by human chorionic gonadotropin in vitro. Mol Hum Reprod 1997, 3:979-984[Abstract/Free Full Text]
  13. Neulen J, Yan Z, Raczek S, Weidel K, Keck C, Weich HA, Marme D, Breckwoldt M: Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/ vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 1995, 6:1967-1971
  14. Lee A, Christenson LK, Patton PE, Burry KA, Stouffer RL: Vascular endothelial growth factor production by human luteinized granulosa cells in vitro. Hum Reprod 1997, 12:2756-2761[Abstract/Free Full Text]
  15. Artini RG, Fasciani A, Monti M, Luisi S, D’Ambrogio G, Genazzani AR: Changes in vascular endothelial growth factor levels and the risk of ovarian hyperstimulation syndrome in women enrolled in an in vitro fertilization program. Fertil Steril 1998, 70:560-564[CrossRef][Medline]
  16. Ferrara N, Chen H, Davis-Smyth T, Gerber HP, Nguyen TN, Peers D, Chisholm V, Hillan KJ, Schwall RH: Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nat Med 1998, 4:336-340[CrossRef][Medline]
  17. Pietrowski D, Keck C: Differential regulation of ANG2 and VEGF-A in human granulose lutein cells by choriogonadotropin. Exp Clin Endocrinol Diabetes 2004, 112:208-214[CrossRef][Medline]
  18. Senger DR, Claffey KP, Benes JE, Perruzzi CA, Sergiou AP, Detmar M: Angiogenesis promoted by vascular endothelial growth factor: regulation through {alpha}1ß1 and {alpha}2ß1 integrins. Proc Natl Acad Sci USA 1997, 94:13612-13617[Abstract/Free Full Text]
  19. Suzuma K, Takagi H, Otani A, Honda Y: Hypoxia and vascular andothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells. Invest Ophthalmol Vis Sci 1998, 39:1028-1035[Abstract/Free Full Text]
  20. Otani N, Minami S, Yamoto M, Shikone T, Otani H, Nishiyama R, Otani T, Nakano R: The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy. J Clin Endocrinol Metab 1999, 84:3845-3851[Abstract/Free Full Text]
  21. Endo T, Kitajima Y, Nishikawa A, Manase K, Shibuya M, Kudo R: Cyclic changes in expression of mRNA of vascular endothelial growth factor, its receptor flt-1 and KDR/flk-1 and Ets-1 in human corpora lutea. Fertil Steril 2001, 76:762-768[CrossRef][Medline]
  22. Makrigiannakis A, Coukos G, Christofidou-Solomidou M, Gour BJ, Radice GL, Blaschuk O, Coutifaris C: N-Cadherin-mediated human granulosa cell adhesion prevents apoptosis: a role in follicular development and luteolysis. Am J Pathol 1999, 154:1391-1406[Abstract/Free Full Text]
  23. Noyes RW, Hertig AT, Rock J: Dating endometrial biopsy. Am J Obstet Gynecol 1975, 122:262-236[Medline]
  24. Remmele W, Hildebrand U, Hienz HA, Klein PJ, Vierbuchen M, Behnken LJ, Heicke B, Scheidt E: Comparative histological, histochemical, immunohistochemical and biochemical studies on oestrogen receptors, lectin receptors, and Barr bodies in human breast cancer. Virchows Arch A Pathol Anat Histopathol 1986, 409:127-147[CrossRef][Medline]
  25. Scott G, Liang H: pp125FAK in human melanocytes and melanoma: expression and phosphorylation. Exp Cell Res 1995, 219:197-203[CrossRef][Medline]
  26. Wayner EA, Orlando RA, Cheresh DA: Integrins {alpha}vß3 and {alpha}vß5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J Cell Biol 1991, 113:919-929[Abstract/Free Full Text]
  27. Meredith JE, Fazeli B, Schwartz MA: The extracellular matrix as a cell survival factor. Mol Biol Cell 1993, 4:953-961[Abstract]
  28. Frisch SM, Francis H: Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 1994, 124:619-626[Abstract/Free Full Text]
  29. Breckwoldt M, Selvaraj N, Aharoni D, Barash A, Segal I, Insler V, Amsterdam A: Expression of Ad4-BP/cytochrome 450 side chain cleavage enzyme and induction of cell death in long term cultures of human granulosa cells. Mol Hum Reprod 1996, 2:391-400[Abstract/Free Full Text]
  30. Makrigiannakis A, Coukos G, Blaschuk O, Coutifaris C: Follicular atresia and luteolysis: evidence of a role for N-cadherin. Ann NY Acad Sci 2000, 900:46-55[Abstract/Free Full Text]
  31. Pankov R, Yamada KM: Fibronectin at a glance. J Cell Sci 2002, 115:3861-3863[Free Full Text]
  32. Aota S, Yamada KM: Fibronectin and cell adhesion: specificity of integrin-ligand interaction. Adv Enzymol Relat Areas Mol Biol 1995, 70:1-21[CrossRef][Medline]
  33. Nicosia RF: What is the role of vascular endothelial growth factor-related molecules in tumor angiogenesis? Am J Pathol 1998, 153:11-17[Free Full Text]
  34. Tamanini C, De Ambrogi M: Angiogenesis in developing follicle and corpus luteum. Reprod Domest Anim 2004, 39:206-216[CrossRef][Medline]
  35. Stouffer RL, Martinez-Chequer JC, Molskness TA, Xu F, Hazzard TM: Regulation and action of angiogenic factors in the primate ovary. Arch Med Res 2001, 32:567-575[CrossRef][Medline]
  36. Wulff C, Wilson H, Wiegand SJ, Rudge JS, Fraser HM: Prevention of thecal angiogenesis, antral follicular growth, and ovulation in the primate by treatment with vascular endothelial growth factor Trap R1R2. Endocrinology 2002, 143:2797-2807[Abstract/Free Full Text]
  37. Fraser HM, Wilson H, Morris KD, Swanston I, Wiegand SJ: Vascular endothelial growth factor trap suppresses ovarian function at all stages of the luteal phase in the macaque. J Clin Endocrinol Metab 2005, 90:5811-5818[Abstract/Free Full Text]
  38. Fraser HM, Dickson SE, Lunn SF, Wulff C, Morris KD, Carroll V, Bicknell R: Suppression of luteal angiogenesis in the primate after neutralization of vascular endothelial growth factor. Endocrinology 2000, 141:995-1000[Abstract/Free Full Text]
  39. Hynes RO: Integrins: versatility, modulation and signaling in cell adhesion. Cell 1992, 69:11-25[CrossRef][Medline]
  40. Wehrle-Haller B, Imhof BA: Integrin-dependent pathologies. J Pathol 2003, 200:481-487[CrossRef][Medline]
  41. Lauffenburger DA, Horwitz AF: Cell migration: a physically integrated molecular process. Cell 1996, 84:359-369[CrossRef][Medline]
  42. Sheetz MP, Felsenfeld DP, Galbraith CG: Cell adhesion: regulation of force on extracellular matrix-integrin complexes. Trends Cell Biol 1998, 8:51-54[Medline]
  43. Coutifaris C, Omigbodun A, Coukos G: The fibronectin receptor {alpha}5 integrin subunit is upregulated by cell-cell adhesion via a cyclic AMP-dependent mechanism: implications for human trophoblast migration. Am J Obstet Gynecol 2005, 192:1240-1255[CrossRef][Medline]
  44. Stupack DG, Cheresh DA: Get a ligand, get a life: integrins, signaling and cell survival. J Cell Sci 2002, 115:3729-3738[Abstract/Free Full Text]
  45. Zhan M, Zhao H, Han ZC: Signaling mechanisms of anoikis. Histol Histopathol 2004, 19:973-983[Medline]
  46. Zhang Z, Vuori K, Reed JC, Ruoslahti E: The {alpha}5ß1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 1995, 92:6161-6165[Abstract/Free Full Text]
  47. Kugu K, Ratts V, Piquette G, Tilly K, Tao XJ, Martimbeau S, Aberdeen GW, Krajewski S, Reed JC, Pepe GJ, Albrecht ED, Tilly JL: Analysis of apoptosis and expression of Bcl-2 gene family members in the human and baboon ovary. Cell Death Differ 1998, 5:67-76[CrossRef][Medline]
  48. Sugino N, Suzuki T, Kashida S, Karube A, Takiguchi S, Kato H: Expression of Bcl-2 and Bax in the human corpus luteum during the menstrual cycle and in early pregnancy: regulation by human chorionic gonadotropin. J Clin Endocrinol Metab 2000, 85:4379-4386[Abstract/Free Full Text]
  49. Vaskivuo TE, Ottander U, Oduwole O, Isomaa V, Vihko P, Olofsson JI, Tapanainen JS: Role of apoptosis, apoptosis-related factors and 17beta-hydroxysteroid dehydrogenases in human corpus luteum regression. Mol Cell Endocrinol 2002, 30:191-200
  50. Chun SY, Billig H, Tilly J, Furuta I, Tsafriri A, Hsueh AJ: Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I. Endocrinology 1994, 135:1845-1853[Abstract]
  51. Dharmarajan AM, Hisheh S, Singh B, Parkinson S, Tilly KI, Tilly JL: Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression. Endocrinology 1999, 140:2555-2561[Abstract/Free Full Text]
  52. Yuan W, Giudice LC: Programmed cell death in human ovary is a function of follicle and corpus luteum status. J Clin Endocrinol Metab 1997, 82:3148-3155[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
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 Google Scholar
Google Scholar
Right arrow Articles by Rolaki, A.
Right arrow Articles by Makrigiannakis, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rolaki, A.