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Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Hiroshi Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: fuji{at}kuhp.kyoto-u.ac.jp.
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Introduction |
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LH is one of the well-known factors promoting human corpus luteum formation, and is considered to regulate angiogenesis by enhancing the secretion of several soluble angiogenic factors, such as vascular endothelial growth factor, fibroblast growth factor, angiopoietin, and angiogenin (1, 2, 3). Recently, in addition to these soluble factors, cell surface molecules mediating cell-cell adhesion have been proposed to play an important role in establishing the new vascular network, participating in the process of arterio-venous anastomosis in embryogenesis and pathological lesions (4). However, there are few reports concerning the system that regulates angiogenesis via direct cell to cell interaction in the ovary.
In recent years it has been demonstrated that Eph receptors and ephrins are membrane-bound molecules that regulate various important cell-cell recognition events during embryonic development, including the formation of spatial boundaries, tissue morphogenesis, and the control of axonal guidance (5, 6, 7). Eph-ephrin interactions have also been reported to regulate neovascularization in both embryonic and adult stages (8, 9).
Ephrins are ligands of Eph receptors that constitute the largest family of receptor tyrosine kinases. Ephrins are all membrane-attached, and this attachment appears to be crucial for their agonistic function. They are subdivided into two subclasses depending on their way of tethering to the cell membrane: five ephrins are linked to the membrane via a glycosyl-phosphatidyl inositol linkage and are referred to collectively as the ephrin A subclass (ephrins A1–A5), whereas three other ephrins have transmembrane and intracellular domains and are referred to as the ephrin B subclass (ephrins B1–B3). Eph receptors are also subdivided into two (A and B) subclasses according to their specificity of binding to ephrins of the A or B subclass (Ephs A1–A8 and Ephs B1–B6, respectively) (10).
Eph-ephrin interactions, which occur at cell to cell interfaces, have been reported to produce a repulsive force between the cells via modification of the cytoskeleton (11) or integrin functions (12, 13, 14). They thereby regulate both cell repulsion and cell attachment to provide information about the movement and position of cells, enabling the cells to move to certain destinations or to form boundaries between two cell populations.
Previously, we reported that integrins are expressed on human luteinizing granulosa cells during corpus luteum formation and suggested that they regulate granulosa cell luteinization in cooperation with their ligands, extracellular matrix proteins (15, 16, 17, 18, 19). As the B class Eph-ephrin system is known not only to mediate cell repulsion by regulating integrin functions, but also to induce endothelial cell migration and arterio-venous anastomosis (8, 9), we speculated that the Eph-ephrin system might be involved in the regulation of luteinization and angiogenesis during corpus luteum formation.
There has been only one report concerning the expression of Eph-ephrin molecules in the mammalian ovary; it showed that ephrin B2 was highly expressed by the endothelium of a subset of new vessels in growing follicles and the corpora lutea of the mouse ovary (20). In the present study we screened the human corpora lutea of the early luteal phase for the expression of B class Eph-ephrin molecules and observed that luteinizing granulosa cells express immunoreactive ephrin B1. Although some mesenchymal cells have been reported to express B class Eph-ephrin proteins that induce sprouting angiogenesis, we have found no published evidence about the status of the Eph-ephrin expression on differentiating cells in neovascularization sites. Therefore, we considered it noteworthy that the differentiating granulosa cells were found to express ephrin B1 during corpus luteum formation. We then used luteinizing granulosa cells isolated from patients undergoing in vitro fertilization treatment to further analyze the cells that expressed the ephrin B1 molecule on their surface.
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Materials and Methods |
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The rabbit antihuman ephrin B1 polyclonal antibody (sc-1011), raised against an internal domain of human ephrin B1, and its blocking peptide (sc-1011P) that was used for immunization were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antihuman Eph B1, B2, and B4 polyclonal antibodies (sc-9319, -1763, and -7284) and the rabbit antihuman ephrin B2 polyclonal antibody (sc-1010) were purchased from Santa Cruz Biotechnology, Inc. The goat antimouse ephrin B1 polyclonal antibody, which was raised against an extracellular domain of mouse ephrin B1 and cross-reacted with human and rat ephrin B1, and recombinant mouse Eph B2-human Fc fusion protein were obtained from Techne Corp. (Minneapolis, MN). The mouse antihuman 3ß-hydroxysteroid dehydrogenase (3ß-HSD) monoclonal antibody was raised in our laboratory. The mouse antihuman aminopeptidase N/CD13 monoclonal antibody (clone MCS-2) was obtained from Nichirei Co. (Tokyo, Japan). The mouse antihuman von Willebrand factor monoclonal antibody (clone F8/86), rabbit antihuman IgG antibody, fluorescein isothiocyanate (FITC)-conjugated swine antirabbit Ig antibody, and FITC-conjugated rabbit antigoat Ig antibody were purchased from Dako (Glostrup, Denmark). The rhodamine-conjugated goat antimouse Ig antibody was obtained from Santa Cruz Biotechnology, Inc. The goat antimouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Samples
Ovarian follicles and corpora lutea formed during the menstrual cycle were obtained from nine women, aged 27–46 yr. These women had undergone unilateral ovarian cystectomy or oophorectomy and contralateral wedge resection for the treatment of benign ovarian tumors. All of the women had a history of regular menstrual cycles (28–32 d), and their ovulatory basal body temperature charts showed normal luteal phase duration. Term placental tissues were obtained from three women after spontaneous normal vaginal delivery. Macroscopically and microscopically normal regions of these tissues were used for this study. Informed consent for the use of these tissue samples was obtained from all patients before the study.
The growing follicles were evaluated morphologically by staining cryosections with hematoxylin and eosin (HE). Follicles obtained during the follicular phase, in which the granulosa cells had normally shaped nuclei, cytoplasm, stratified layers, and mitotic figures, were classified as growing or preovulatory follicles. Follicles that were irregularly shaped, showed blood cell invasion, and lacked mitotic figures were classified as atretic follicles (21). The postovulatory date of the corpus luteum was evaluated according to the histological dating system described by Corner (22), using HE-stained sections of 10% formalin-fixed and paraffin-embedded samples.
Isolation of human luteinizing granulosa cells
Human luteinizing granulosa cells were isolated from patients, aged 33–40 yr, who had undergone in vitro fertilization therapy as previously reported (17). Briefly, administration of a GnRH agonist (buserelin acetate, Aventis Pharma Co., Tokyo, Japan) was initiated in the midluteal phase or the early follicular phase. All patients subsequently received pure FSH (Serono Japan, Co., Tokyo, Japan) or human menopausal gonadotropin (Organon Japan Co., Tokyo, Japan) from cycle d 3 for ovarian stimulation until the dominant follicle reached a diameter of more than 18 mm, followed by an injection of human chorionic gonadotropin (Mochida Pharmaceutical Co., Osaka, Japan) 36 h before oocyte retrieval. The follicular fluid was collected and centrifuged. The suspension of granulosa cells was overlaid onto Ficoll-Hypaque (Nacalai Tesque, Kyoto, Japan) and centrifuged at 400 x g for 25 min. The cells were then collected from the interphase and resuspended in RPMI 1640 culture medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). The cells were collected after mild washing and suspended in culture medium at a density of 3 x 105 cell/ml.
Indirect immunohistochemical staining of frozen sections
Indirect immunofluorescence histochemistry was performed as previously described (16). Three preovulatory follicles and six corpora lutea in the early luteal phase were examined. Each specimen was embedded in OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL), snap-frozen in liquid nitrogen, and stored at -80 C. The frozen tissues were sliced at 6-µm thickness using a cryostat microtome (Cryocut 1800, Reichert-Jung, Heidelberg, Germany), immediately air-dried on aminopropyltriethoxysilane-coated glass slides (Matsunami Glass, Osaka, Japan), and then fixed in acetone at -20 C for 5 min.
The slides were incubated with rabbit antihuman ephrin B1 or B2 polyclonal antibody or with goat antihuman Eph B1, B2, or B4 polyclonal antibody (20 µg/ml) for 40 min at room temperature. After washing in PBS, they were incubated with FITC-conjugated swine antirabbit Ig antibody or FITC-conjugated rabbit antigoat Ig antibody (diluted 1:40) for 40 min at room temperature in the dark. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA), which reduces fluorescence fading, and examined under a fluorescence microscope (Nikon, Tokyo, Japan). Serial cryosections were also stained with HE after acetone fixation.
To confirm the specificity of the primary antiephrin B1 polyclonal antibody, it was substituted with the same primary antibody that had been preincubated with a 5-fold excess of its immunizing peptide for 2 h at room temperature. As a control, the primary antibody was also preincubated with the same amount of Eph A5-immunizing peptide (Santa Cruz Biotechnology, Inc.).
In the double immunohistochemical stainings, 3ß-HSD and aminopeptidase N were used as markers for steroidogenic cells and thecal cells, respectively (23). von Willebrand factor was used as a marker for endothelial cells. After the reaction with the antiephrin B1 antibody and with the subsequent FITC-conjugated secondary antibody, the slides were incubated with antihuman von Willebrand factor antibody (5 µg/ml), antihuman 3ß-HSD antibody (5 µg/ml), or antihuman aminopeptidase N antibody (5 µg/ml) for 40 min at room temperature. After washing twice, they were incubated with rhodamine-conjugated goat antimouse Ig antibody (diluted 1:40) for 40 min at room temperature in the dark. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium, and then examined under a confocal laser scanning microscope (Carl Zeiss, Inc., Jena, Germany).
Flow cytometry
The isolated luteinizing granulosa cells were washed in Hanks’ balanced salt solution (HBSS) and sedimented by centrifugation. After the supernatant was removed, the cells were incubated with 10 µl goat antimouse ephrin B1 polyclonal antibody (Techne Corp.; 50 µg/ml) or goat antimouse IgG antibody (50 µg/ml) as a negative control for 30 min at 4 C. After washing in HBSS, the cell pellet was incubated with 20 µl FITC-conjugated rabbit antigoat Ig antibody (diluted 1:20) for 30 min at 4 C in the dark. After washing in HBSS, the cells were resuspended in the same solution, and viable cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences, Tokyo, Japan). The negative level of relative fluorescence intensity was judged from the histogram of the control group stained with goat antimouse IgG antibody in each experiment. The positive area in the cells stained with antimouse ephrin B1 polyclonal antibody was determined in the right side from the above intensity level. The fluorescence intensity of 5000 viable cells that were gated as luteinizing granulosa cells by cell size and granularity was evaluated in each flow cytometric experiment. After the incubation with secondary antibody, the washed cells were also suspended in PBS/glycerin (1:1, vol/vol) and observed under a fluorescence microscope. These experiments were repeated four times.
RNA isolation
Human corpus luteum tissues were immediately frozen in liquid nitrogen and stored at -80 C until RNA extraction. The total RNAs derived from these tissues and from isolated granulosa cells were extracted using a commercial kit (TRIzol, Life Technologies, Inc.). The total RNAs were also prepared from the three tissue samples of human term placentas.
RT-PCR analysis of mRNA expression for Eph B receptors and B ephrins in the human corpora lutea
Five micrograms of total RNAs from corpora lutea of the early luteal phase (postovulatory d 2–4; n = 5) were reverse transcribed with random primers using a commercial kit (First Strand cDNA Synthesis Kit, Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification. The primers for human Eph B receptors, B class ephrins, and S26 that were designed based on the reported nucleotide sequence of the respective human proteins are listed in Table 1
(24, 25, 26, 27, 28, 29, 30, 31, 32). We did not perform RT-PCR for Eph B5 receptor because the nucleotide sequence of human Eph B5 had not been reported. After PCR amplification, 10 µl of each PCR product was electrophoresed in 1% agarose gels, and amplified cDNA bands were visualized by ethidium bromide staining. After cloning the PCR products and verifying their sequences, the enzymatically digested cDNA insert corresponding to the human ephrin B1 gene was purified and used as a probe for subsequent Northern blot analysis.
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Ten micrograms of total RNAs derived from isolated human luteinizing granulosa cells (n = 5), corpora lutea of the early luteal phase (postovulatory d 2–4; n = 5), and term placenta tissues (n = 3) were separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, Arlington, IL). The membrane was incubated with prehybridization solution (Rapid Hybridization Buffer, Amersham Pharmacia Biotech,) for 30 min at 65 C and then hybridized with the 32P-labeled ephrin B1 cDNA probe for 2 h at 65 C in the same solution. After hybridization, the membrane was washed in 2x standard saline citrate (2 mM sodium citrate and 20 mM sodium chloride in distilled water, pH 7.0) with 0.1% sodium dodecyl sulfate at room temperature for 15 min, and then in 0.2x standard saline citrate with 0.1% sodium dodecyl sulfate at 65 C for 30 min. Afterward the membrane was subjected to autoradiography and then washed and rehybridized with the S26 probe to correct for the amount of loaded RNA.
Binding assay of preclustered recombinant Eph B2-human Fc fusion protein to granulosa cells
Recombinant mouse Eph B2-human Fc fusion protein was clustered by incubation with rabbit antihuman IgG antibody (5 µg antihuman IgG for 50 µg Eph B2-Fc protein in 1 ml sterile PBS) for 2 h at 4 C. Then the isolated granulosa cells were incubated with the preclustered Eph B2-Fc at a concentration of 500 ng/ml in culture medium at 37 C for 1 h. The cells were collected by centrifugation, washed in HBSS, and reacted with FITC-conjugated swine antirabbit Ig antibody for 30 min at 4 C to detect the preclustered Eph B2-Fc that had bound to the cell surface of granulosa cells. As negative controls, the cells were also incubated with 1) Eph B2-Fc protein and FITC-conjugated swine antirabbit Ig antibody in the absence of rabbit antihuman IgG antibody, 2) rabbit antihuman IgG antibody and FITC-conjugated swine antirabbit Ig antibody in the absence of Eph B2-Fc protein, or 3) only FITC-conjugated swine antirabbit Ig antibody. After washing, the reacted cells were suspended in PBS/glycerin (1:1, vol/vol) and examined under a fluorescence microscope. In some cases the cells were suspended in HBSS and were subjected to flow cytometry. Binding was assessed by histograms. These binding assays were repeated five times.
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Results |
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For the purpose of screening, we performed RT-PCR analysis using mRNA samples derived from corpus luteum tissues of the early luteal phase (n = 5). The cDNA products corresponding to Eph B1, Eph B2, Eph B4, ephrin B1, and ephrin B2 were detected by ethidium bromide staining (Fig. 1). The DNA sequences of individual products were verified by DNA sequencing to confirm their identities with reported ones (24, 25, 27, 29, 30, 32). The mRNAs of Eph B3, Eph B6, and ephrin B3 were not detected (Fig. 1).
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To examine the expression profiles of Ephs and ephrins detected by RT-PCR analysis in human corpora lutea of the early luteal phase, we performed immunohistochemical analysis using available polyclonal antibodies. Among these antibodies, antiephrin B1 antibody worked well for detecting the immunolocalization of ephrin B1 protein in preovulatory follicles (n = 3; Figs. 2 and 3) and corpora lutea of the early luteal phase (n = 6; Figs. 4 and 5). Unfortunately, we did not detect immunoreactive ephrin B2 or Eph B1, B2, or B4 protein on the ovarian frozen sections with the antibodies employed in this study.
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). The expression of ephrin B1 was not observed on the von Willebrand factor-bearing endothelial cells surrounding the basement membrane in the theca interna cell layer (Fig. 3).
In contrast, after ovulation, a rapid increase in ephrin B1 expression was observed on the luteinizing granulosa cells of corpora lutea. On postovulatory d 2, the loose radial arrangement of the granulosa persisted, but luteinization was advancing rapidly (22). The expression of ephrin B1 was clearly observed on luteinizing granulosa cells (Fig. 4).
Granulosa and theca interna cells are indistinguishable because of their luteinization (22). Using a marker for theca interna cells, aminopeptidase N (23), the expression of ephrin B1 on luteinizing theca interna cells was shown to decrease (Fig. 5, A–C).
All of these positive immunostainings were diminished by preincubation of the primary polyclonal antibody with the synthetic peptide that had been used as the ephrin B1 antigen for immunization of rabbits (Figs. 2C and 4D). However, these immunostainings were not inhibited by preincubation with the immunizing peptide for Eph A5 (data not shown).
On postovulatory d 4, the cytoplasm of the granulosa cells showed light lipid stippling, and the pale-staining nuclei represented only about one third of the cell volume. The nuclei of the theca cells represented a little more than one third of the cell volume, and theca interna began to be distinguishable from the granulosa cells (22). The expression of ephrin B1 was clearly observed on luteinizing granulosa cells (Fig. 5D). To identify the steroid hormone-producing cells, we performed double staining of luteal cells using anti-3ß-HSD antibody. All of the ephrin B1-positive cells were also stained by anti-3ß-HSD antibody (Fig. 5, E and F). Conversely, ephrin B1 was not detected on the 3ß-HSD-negative cells. On the other hand, the expression of ephrin B1 on the luteinizing theca interna cells, which were positive for aminopeptidase N (23), was hardly detected (data not shown).
Cell surface expression of ephrin B1 on isolated human luteinizing granulosa cells detected by flow cytometry
Flow cytometric analysis clearly demonstrated the cell surface expression of ephrin B1 on isolated human luteinizing granulosa cells (Fig. 6). The mean positive rate was 56.2 ± 4.9% (n = 4; mean ± SE).
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Northern blot analysis showed single mRNA bands of 3.5 kbp corresponding to ephrin B1, a size comparable with that shown in a previous report (29), in the mRNA samples derived from freshly isolated human luteinizing granulosa cells (n = 5), human corpora lutea in the early luteal phase (n = 5), and human term placentas (n = 3) used as positive controls (29) (Fig. 7).
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To evaluate the biological reactivity of ephrin B1 expressed on luteinizing granulosa cells, we examined the binding of Eph B2 to freshly isolated granulosa cells using recombinant Eph B2-human Fc fusion protein. When the granulosa cells were reacted with the recombinant Eph B2-human Fc fusion protein that had been clustered by rabbit antihuman IgG antibody and then incubated with FITC-conjugated antirabbit IgG antibody, the binding of Eph B2-Fc fusion protein to the granulosa cell surface was clearly detected by microscopic observation and flow cytometry (Fig. 8). In the three control groups, no staining was observed.
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Discussion |
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Using antibodies, we observed immunolocalization of ephrin B1 in the human ovary. Ephrin B1 was expressed on luteinizing granulosa cells, but was hardly detected on granulosa cells in preovulatory follicles. When this antibody was preincubated with the synthetic peptide that had been used for immunization, the positive immunohistochemical staining disappeared, indicating the specificity of the immunostaining data. We also examined ephrin B1 expression on human luteinizing granulosa cells isolated from patients undergoing in vitro fertilization therapy. The cell surface expression of ephrin B1 on these luteinizing granulosa cells was further confirmed by flow cytometry using another antiephrin B1 polyclonal antibody. In addition, the expression of ephrin B1 mRNA of a size comparable with that previously reported was confirmed by Northern blot analysis of RNA samples derived from both isolated luteinizing granulosa cells and the corpora lutea of the early luteal phase. Furthermore, immunohistochemical double staining using anti-3ß-HSD or antiaminopeptidase N monoclonal antibody showed that the ephrin B1-expressing cells in the corpora lutea of the early luteal phase also expressed 3ß-HSD, but hardly expressed aminopeptidase N, indicating that ephrin B1-expressing cells are steroid hormone-producing cells that belong mainly to the granulosa cell lineage. Taken together with the immunohistochemical study results in preovulatory follicles, the finding that ephrin B1 expression on granulosa cells seemed to increase rapidly after the LH surge or ovulation suggests its involvement in corpus luteum formation.
During corpus luteum formation, luteinizing granulosa cells and migrating endothelial cells have to communicate with each other to establish a fine vascular network around fully luteinized, large luteal cells of the mature corpus luteum. Recently, it was demonstrated that clustered ephrin B1 promoted endothelial capillary-like assembly in an in vitro assay of angiogenesis (35). It is well known that vascular endothelial cells communicate with each other via B class Eph-ephrin interactions during vascular development and that these interactions are especially essential in arterio-venous anastomosis (8, 9, 36, 37, 38). In addition, vascular endothelial cells were reported to express Eph B3 and Eph B4, thereby enabling their angiogenic interactions with the adjacent mesenchymal cells, which express B class ephrins as well (8, 38, 39). Thus, the Eph-ephrin system is considered to play an important role in the cell to cell interactions between endothelial cells and mesenchymal cells during neovascularization (39). Taking these previous reports into consideration, it can be speculated that the luteinizing granulosa cells that express ephrin B1 may directly communicate with the vascular endothelial cells through Eph-ephrin interactions to induce angiogenesis.
In this study we demonstrated the mRNA expression of several Eph B receptors, such as Eph B1, Eph B2, and Eph B4, in early luteal phase human corpora lutea by RT-PCR analysis. Although we failed to confirm the precise immunohistochemical localization of the Eph B receptor molecule using the available antibodies, our findings suggest that Eph B receptor-expressing cells are present and can interact with the ephrin B1-expressing luteinizing granulosa cells in the corpora lutea during corpus luteum formation.
To investigate the ability of human luteinizing granulosa cells to interact with Eph B-expressing cells, we examined whether granulosa cells have the ability to bind to Eph B molecules. As ephrin B1 was reported to bind to Eph B2 receptor, we used recombinant Eph B2-Fc fusion protein. The activation of Eph-ephrin signaling is known to require membrane attachment or artificial clustering of the ligands (10, 40). Therefore, to examine the biological reactivity with B class ephrin(s) on luteinizing granulosa cells, Eph B2-Fc fusion protein was preclustered by incubation with rabbit antihuman IgG antibody and then reacted with granulosa cells. The distribution of the Eph B2-Fc fusion protein on the cell surface was clearly detected by means of immunocytochemical microscopic observation and flow cytometry. No staining was observed in the incubations with negative controls. These findings suggest that B class ephrin(s) expressed on the granulosa cells binds to Eph B-bearing cells during corpus luteum formation and therefore support our speculation that luteinizing granulosa cells may directly communicate with vascular endothelial cells through Eph-ephrin interactions.
Notably, Eph-ephrin interaction has been demonstrated to induce bidirectional signaling into both the Eph- and ephrin-bearing cells (41). In other words, when ephrin B1-expressing cells were challenged with clustered Eph B2-Fc fusion protein, the cell surface ephrin B1 became phosphorylated on its tyrosine residues in the cytoplasmic domains, and the reverse signal was transduced into the ligand (ephrin B1)-expressing cells (11, 41, 42). Therefore, the binding experiments employed in the present study may have induced intracytoplasmic activation in the ephrin-expressing granulosa cells. Our preliminary experiments failed to demonstrate a significant effect of Eph B2-Fc fusion protein on progesterone production by granulosa cells (data not shown), but in future studies we will further examine the possibility that this interaction induces some other cellular responses in granulosa cells in vivo.
The results of our analysis of mRNA expression by RT-PCR indicate that it is possible that luteinizing granulosa cells express not only B-class ephrins, but also Eph B receptors. Assuming that the luteinizing granulosa cells express both Eph B receptor(s) and B-class ephrins, one of the expected effects of their activation is the repulsion between them. During the first few days after ovulation, the luteinizing granulosa cells have loose contacts with each other, which allow endothelial cells to migrate among them while they are expanding during the progression of luteinization. A much faster formation of the tight connections around luteinizing granulosa cells would be unfavorable for tissue remodeling. Therefore, the programmed regulation of cell to cell spacing may be necessary for corpus luteum formation. To maintain appropriate cell to cell spacing, the coordinated balance of two main forces (an adhesive force and a repulsive one) would be required. The repulsive force might be produced via modification of the cytoskeleton (11) or integrin functions (12, 13, 14). We previously reported that the luteinizing granulosa cells express integrins and control their luteinization via integrin-extracellular matrix interactions. Recently, the Eph B2-ephrin B1 interaction was reported to regulate integrin functions to induce cell detachment (13), suggesting that this interaction may regulate repulsive forces among cells. Taking all these facts together, it is reasonable to consider that Eph and ephrin molecules may be involved in the regulation of appropriate cell to cell spacing among luteinizing granulosa cells by exerting a repulsive force that counterbalances other adhesive forces.
In conclusion, this study showed that ephrin B1 was expressed on human luteinizing granulosa cells during corpus luteum formation. These cells were also shown to have the ability to bind to Eph B molecules, suggesting that they interact with Eph B-expressing cells. As B class Eph-ephrin interactions are well known to mediate endothelial migration and cell to cell repulsion, we speculate that the Eph-ephrin system might be one of the regulators of tissue remodeling during corpus luteum formation, especially of the formation of the new vascular network.
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Acknowledgments |
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Footnotes |
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Abbreviations: FITC, Fluorescein isothiocyanate; HBSS, Hanks’ balanced salt solution; HE, hematoxylin and eosin; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase.
Received December 4, 2002.
Accepted May 5, 2003.
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vß3 and 5ß1 integrins. EMBO J 18:2165–2173[CrossRef][Medline]
6 is a differentiation antigen of human granulosa cells. J Clin Endocrinol Metab 80:2899–29056ß1. J Clin Endocrinol Metab 82:2122–21285 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 3:979–9842 and collagen type IV: possible involvement of collagen type IV in granulosa cell luteinization. Mol Hum Reprod 5:607–617This article has been cited by other articles:
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