Molecular Human Reproduction, Vol. 5, No. 10, 920-926,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of ovarian function |
Co-expression of integrin-associated protein (IAP/CD47) and its ligand thrombospondin-1 on human granulosa and large luteal cells
1 Department of Gynecology and Obstetrics and 2 Clinical Molecular Biology, Faculty of Medicine, and 3 Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto 606–01, Japan
Abstract
In the present study, we have raised a monoclonal antibody (mAb) designated OG-4 against human granulosa cells (GC). By immunohistochemistry, the expression of OG-4 antigen was observed on human GC and large luteal cells, but not on thecal and small luteal cells. A complementary DNA (cDNA) clone encoding OG-4 antigen was screened and isolated by a panning method using OG-4 mAb from a human corpus luteum (CL) cDNA library that was expressed transiently in COS-7 cells. Nucleotide sequencing revealed that OG-4 antigen was identical to integrin-associated protein (IAP)/CD47 antigen. Subsequent reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that the isoform 2 mRNA of IAP is predominantly expressed in human GC and CL. The expression of thrombospondin-1 (TSP-1), which is a ligand for IAP, was also observed in human GC by immunocytochemistry and RT–PCR. Co-expresson of IAP and TSP-1 on human GC may suggest that TSP-1 has a physiological role in GC function possibly via IAP in an autocrine fashion.
granulosa cells/integrin-associated protein/monoclonal antibody/panning/thrombospondin-1
Introduction
In order to investigate the regulatory factors in ovarian physiology, we raised several monoclonal antibodies (mAb) in mice by immunizing them against human granulosa cells (GC). One of these mAb (OG-1), which reacted with the cell surface molecule of human GC, was shown to recognize integrin 
6, by analysis of partial amino acid sequence of the purified antigen (Fujiwara et al., 1993
; Honda et al., 1995). Laminin, which is a ligand for integrin 6ß1, was shown to suppress GC luteinization via integrin 6ß1 (Fujiwara et al., 1997). In addition, integrin 2 and 5 were found to be expressed on luteinizing GC, suggesting the involvement of integrins in GC function (Honda et al., 1997; Yamada et al., 1999).
In this study, we raised a new mAb, named OG-4, against human GC. This mAb reacted with the cell surface molecule of human GC and large luteal cells. Cloning and sequencing of a complementary DNA (cDNA) encoding OG-4 (clone 6) antigen using a mammalian cell expression vector and immunoselection (Aruffo and Seed, 1987) revealed that OG-4 antigen was identical to integrin-associated protein (IAP)/CD47 antigen (Lindberg et al., 1993).
IAP was firstly identified as a membrane protein which is associated with vß3 integrin (Brown et al., 1990; Lindberg et al., 1993). IAP is also present on lymphocytes that express little vß3 integrin (Brown et al., 1990), and the association of IAP with integrin 2ß1 is suggested in human platelets (Brown et al., 1990; Dorahy et al., 1997). IAP is reportedly expressed on normal adult tissues (Mawby et al., 1994), and the expression of IAP mRNA in mouse ovarian tissue has been previously detected by reverse transcription–polymerase chain reaction (RT–PCR) analysis (Reinhold et al., 1995). In addition, IAP is also identical to OA3 antigen, initially thought to be specific for ovarian carcinoma cells (Cambell et al., 1992). However, the precise expression profiles of IAP in the normal human ovary have not been reported. Recent studies demonstrated that IAP is a receptor for TSP-1 (Gao et al., 1996; Dorahy et al., 1997), which is a matrix glycoprotein implicated in the regulation of the motility, proliferation, and differentiation of many cell types (Frazier, 1991; Bornstein and Sage, 1994). Therefore, we examined the expression of TSP-1 as well as IAP in the human ovary.
Materials and methods
Cell culture
COS-7 cell was obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium/10% calf serum.
Human ovaries
The growing follicles (n = 13) and pre-ovulatory follicles (n = 8), corpora lutea (CL) of the early (n = 10), mid-luteal (n = 12) and late luteal phases (n = 8) were obtained from 51 women, aged between 22 and 46 years old. They had undergone unilateral ovarian cystectomy or oophorectomy and contralateral wedge resection to treat benign ovarian tumours. All women had a history of regular menstrual cycles (28–30 days) and their ovulatory basal body temperature charts were of normal luteal phase length. CL of pregnancy were obtained from 10 pregnant patients aged from 26 to 46 years, who had undergone hysterectomy at 6 (n = 2), 7, 8, 9, 10, 13 (n = 2), 14 and 15 weeks gestation due to uterine myoma and/or cervical cancer. In all patients, fetal growth was normal on ultrasonographic examination. For RNA isolation, CL was immediately frozen in liquid nitrogen and stored at –80°C. For immunohistochemistry, each specimen was embedded in OCT compound (Tissue-Tec; Miles Scientific, Naperville, IL, USA), snap-frozen in liquid nitrogen, and stored at –80°C. Informed consent was obtained from each patient. Follicles were morphologically evaluated by haematoxylin and eosin (HE)-stained cryosections or the HE-stained sections from the identical samples that were fixed with 10% formalin and embedded with paraffin. Follicles obtained in the follicular phase, in which GC had regular-shaped nuclei, cytoplasm, stratified layers and mitotic figures, were classified as growing or pre-ovulatory follicles (Ryan, 1981). The CL day was re-evaluated according to histological dating, using HE-stained tissue sections (Corner, 1956).
Isolation of human luteinizing granulosa cells
Fresh human GC were obtained from patients aged from 27 to 41 years old who had undergone in-vitro fertilization (IVF) treatment as previously described (Fujiwara et al., 1994). The aspirated follicular fluid was centrifuged, and the resuspended GC were overlaid on Ficoll-Hypaque and centrifuged at 400 g for 30 min. GC were collected from the interphase, washed twice with phosphate-buffered saline (PBS) and were subjected to mAb production, immunocytochemistry, or RNA isolation.
Production and selection of mAb
The production and selection of mAb were performed as described previously (Fujiwara et al, 1993). Briefly, 8 week old BALB/c mice were immunized with human GC obtained as described above. After the spleen cells of the mice were fused with X63Ag8 myeloma cells, using polyethylene glycol 4000 (Köhler and Milstein, 1975), the cells were cultured in 96-well microtitre plates with HAT medium [Roswell Park Memorial Institute 1640 medium (Flow Lab., Irvine, UK) containing 15% fetal bovine serum (Flow Lab., Mclean, VA, USA), 0.1 mmol/l hypoxanthine, 0.4 mol/l aminopterin, and 16 mmol/l thymidine]. Supernatants from the growing hybridomas were screened by performing indirect immunofluorescence staining of 1 day cultured human GC obtained from the patients who had had IVF treatment, and then by such staining of frozen ovarian sections. Hybridomas of interest were cloned twice by the limiting dilution method. The Ig isotype of the culture supernatants from positive clones was determined using an isotyping kit for mouse mAb (Serotec, Oxford, UK). The positive hybridoma clones were expanded and injected intraperitoneally into female mice previously treated with pristane (2,6,10,14-tetramethylpentadecane; Aldrich, Milwaukee, WI, USA). IgG was purified from ascitic fluid with Affi-gel protein A (Bio-Rad Labs, Richmond, CA, USA).
Immunohistochemical examination of OG-4 antigen expression in human ovaries
Indirect immunofluorescence histochemistry was performed as previously described (Fujiwara et al., 1992). Frozen tissues were sliced to 7 µm thickness using a cryostat microtome (Cryocut 1800; Reichert-Jung, Heidelberg, Germany), immediately air-dried on Neoplane (Nisshin EM, Tokyo, Japan)-coated glass slides, and fixed in acetone at –20°C for 5 min. The slides were incubated with OG-4 mAb (5 µg/ml, diluted in culture medium) or anti-trinitrophenyl (TNP) mouse mAb (Tsujimura et al., 1990) (negative control, IgG1 class, 5 µg/ml) for 40 min at room temperature. After washing in PBS, they were incubated with the fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse Ig antibody (diluted 1:40, Dakopatts, Glostrup, Denmark) for 40 min at room temperature in the dark. The slides were washed, mounted with mounting agent (Perma Fluor Aqueous Mounting Medium; Immunon, Pittsburgh, PA), and examined under a fluorescent microscope (Nikon, Tokyo, Japan). The cryosections were also stained with anti-IAP/CD47 mAb (5 µg/ml, BRIC126; Dainippon Pharmaceutical Co. Ltd, Osaka, Japan).
Indirect immunofluorescence staining of isolated GC
Isolated GC (2x105) were incubated at 4°C for 30 min with OG-4 mAb (100 µg/ml), mAb against human TSP-1 (clone 133, class IgG1, 100 µg/ml; Genzyme, Cambridge, MA, USA), or with anti-TNP mAb (100 µg/ml). The cells were washed twice with Hanks' balanced salt solution and incubated with FITC-conjugated rabbit anti-mouse Ig (diluted to 1:40) at 4°C for 30 min in the dark. Cells were then washed twice, resuspended in glycerin/PBS (1:1), mounted on glass slides, and examined with a fluorescence microscope.
cDNA library construction
Poly(A)+ RNA was prepared from human CL by oligo (dT) spin columns of total RNA isolated by the TRIzol method (Gibco BRL, Rockville, MD, USA). cDNA was synthesized by priming with oligo (dT) primer using a SuperScript Choice system (Gibco BRL) and ligated with BstXI adaptor (Invitrogen, Carlsbad, CA, USA). cDNA was ligated into a BstXI-digested pME18S mammalian cell expression plasmid vector, which carries a strong chimeric promoter of SV40 and SR (Takebe et al., 1998), and ligated plasmid DNA was transformed into Escherichia coli DH-5 by electroporation (Cell Porator System; Gibco BRL). The cDNA library obtained contained 1x106 clones.
Expression cloning of OG-4 antigen cDNA
The cDNA library was induced into subconfluent COS-7 cell cultures in 10 cm dishes by the DEAE-dextran transfection method. Detachment, treatment of transfected COS-7 cells with OG-4 mAb, and recovery of plasmid DNA were carried out as previously described (Aruffo and Seed, 1987) except that magnetic beads rather than antibody-coated dishes were used for recovery of OG-4 expressing COS-7 cells. Briefly, the OG-4 mAb-treated cells were resuspended in 1 ml PBS containing 0.5 mM EDTA, 0.02% sodium azide and 5% fetal bovine serum. Fifty µl of sheep anti-mouse IgG-coated magnetic beads (Dynabeads; Dynal A. S., Oslo, Norway) were added, and the cells were incubated on ice for 30 min with periodic gentle shaking. OG-4-expressing cells coated with magnetic beads were recovered by several washes in PBS containing EDTA and fetal bovine serum using a magnetic particle concentrator according to the recommendations of the manufacturer (Dynal). Episomal plasmid DNA was collected from cells bound to magnetic beads by the Hirt procedure (Hirt, 1967) and transformed into E.coli. The resulting colonies were pooled and amplified in liquid culture, and isolated plasmid DNA was subjected to the next cycle of the DEAE–dextran transfection. After five rounds of transfection, individual plasmid clones were transfected into COS-7 and cells were stained with OG-4 mAb as described above. The cDNA insert was digested with XhoI, cloned into pBluescript SK (–), and verified by sequencing (Higuchi et al., 1995a).
RNA isolation and RT–PCR
Total RNA from human GC or CL were isolated as described above, and 2 µg of total RNA from each sample were reverse-transcribed with random primers by a commercial kit (First Strand cDNA Synthesis Kit; Pharmacia, Inc., Piscataway, NJ, USA). The resulting cDNA mixture was subjected to 30 cycles of PCR amplification with oligonucleotides from human IAP/CD47 (Lindberg et al., 1993) (sense primer 5'-CCTATATCCTCGCTGTGGTT-3': position 882–901, anti-sense primer 5'-ACTTTTCTTGTTTCTTCTCC-3': position 1131–1150), TSP-1 primers (Hennessy et al., 1989) (sense primer 5'-TCCCCGTGGTCATCTTGTTC-3': position 1435–1454, anti-sense primer 5'-TAGTTGCACTTGGCGTTCTT-3': position 2067–2086), or with primers for constitutively expressed S26 ribosomal protein cDNA (Higuchi et al., 1995b). PCR products were run on 1% agarose gel or 4% gel and stained with 1 µg/ml ethidium bromide. Each PCR product was cloned and sequenced as described above.
Results
OG-4 antigen expression in human ovarian follicles and CL
A murine mAb designated OG-4 (IgG1 isotype) was generated by immunizing mice with human GC obtained from patients who underwent oocyte retrieval during an IVF programme.
In the growing follicles of 2 and 3 mm (n = 2), 4 mm (n = 2), 5 mm (n = 2), 6 and 7 mm, 8, 11, 14 and 16 mm in diameter and pre-ovulatory follicles (18–20 mm, n = 8), high expression of OG-4 antigen was detected on GC by immunohistochemical staining (Figures 1 and 2). Theca interna and stromal cells did not express OG-4 antigen. The intensity of OG-4 antigen expression on GC was constant throughout follicular development.
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In the menstrual and pregnant CL, OG-4 antigen was highly expressed on large luteal cells (Figure 3
). The expression of OG-4 antigen was not detected on small luteal and stromal cells. These expression profiles are summarized in Table I.
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Immunocytochemical staining showed the cell surface expression of OG-4 antigen on the isolated human GC (Figure 4
).
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Identification of OG-4 antigen as IAP/CD 47
cDNA clones encoding the OG-4 antigen were recovered from a CL cDNA library by antibody panning of transfected COS-7 cells. After five successive rounds of antibody panning and plasmid retrieval, 10 plasmid clones were recovered for further analysis. One clone, clone OG-4–6, showed the cell surface expression of OG-4 antigen on the COS-7 cells as detected by indirect immunofluorescence (Figure 5A
). Digestion of this clone with XhoI and agarose gel electrophoresis showed the inserts of 1.2 kb in clone OG-4–6.
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Plasmid clone OG-4–6 was subjected to DNA sequencing, and comparison of the nucleotide sequences with the EMBL/GenBank databases showed that clone OG-4–6 was identical to the human IAP/CD 47 (Lindberg et al., 1993
). The profiles of immunohistochemical staining of ovarian sections with anti-IAP/CD 47 mAb (BRIC126) were very similar to those with OG-4 mAb (data not shown).
Alternatively spliced forms of human IAP mRNA in GC and CL
To determine the expression of alternatively spliced forms of IAP, RT–PCR on human GC and non-pregnant CL was carried out using oligonucleotides which were present in all four forms of IAP and which bracketed the region of the mRNA encoding the alternatively spliced cytoplasmic tails. Judging from the length of PCR products and subsequent sequencing, the isoform 2 mRNA of IAP was revealed to be predominantly expressed in freshly isolated human GC and in the CL of menstrual cycle (Figure 6; upper panel) and pregnancy (data not shown).
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Expression of TSP-1 in GC and CL
First strand cDNA from isolated human GC and non-pregnant CL were subjected to RT–PCR analysis using TSP-1 primers. Expression of TSP-1 mRNA was detected in isolated GC and in the CL of menstrual cycle (Figure 6
; middle panel) and pregnancy (data not shown).
Immunocytochemical staining also detected TSP-1 protein on the cell surface of isolated human GC (Figure 7A).
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Discussion
In the present study, we raised a mAb designated OG-4, which reacted with the cell surface molecule of human GC. By immunohistochemical analysis, the expression of OG-4 antigen was observed on human GC and large luteal cells but not on theca interna and small luteal cells. This expression profile of OG-4 antigen is compatible with our previous studies which demonstrated that human large luteal cells including those in CL of pregnancy are derived from GC (Fujiwara et al., 1992, 1993, 1996). We constructed a cDNA library derived from human CL to identify a cDNA clone which encodes OG-4 antigen. The panning analysis of transient expression of CL cDNA library in COS-7 cells, one cDNA clone, OG-4–6, was revealed to encode OG-4 antigen. DNA sequencing of this clone OG-4–6 demonstrated that OG-4 antigen is identical to IAP/CD47.
IAP is a unique integrin family member with an amino-terminal integrin variable domain, a multiple membrane-spanning domain, and a short alternatively spliced carboxy-terminal cytoplasmic tail (Lindberg et al., 1993). Although IAP is widely expressed, the tissue-restricted distribution of the various isoforms suggests that IAP might have different functions in different tissues dependent on the nature of its cytoplasmic tail (Reinhold et al., 1995). We therefore carried out RT–PCR in order to examine the isoforms of IAP mRNA expressed in human ovary, and demonstrated that GC and CL predominantly express form 2 mRNA of IAP. This observation is consistent with the previous study which reported the abundant expression of form 2 IAP mRNA in mouse ovarian tissue (Reinhold et al., 1995). These four cytoplasmic tails are reported to be highly conserved between mouse and human (Reinhold et al., 1995), suggesting the essential role of form 2 cytoplasmic tail in ovarian IAP function.
TSP-1 is the most abundant protein of platelet alpha granules. Once platelets release the granules, the secreted TSP-1 is reported to bind to IAP on the surface of platelets and activate the platelet integrin IIbß3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase (Chung et al., 1997). In the present study, we have demonstrated that the isolated human GC express TSP-1 mRNA as well as IAP mRNA. Subsequent immunocytochemical analysis demonstrated that TSP-1 protein is present on the cell surface of isolated GC. The co-expresson of TSP-1 and IAP on human GC suggests that TSP-1 exerts some physiological effect on GC function via IAP in an autocrine fashion. Dreyfus et al. reported that rat GC express TSP-1 and its membrane receptor. They also demonstrated that TSP binding to GC was not inhibited by GRGDS peptides, which suggests that TSP binding does not depend on the RGD sequence (Dreyfus et al., 1992). This finding is compatible with our speculation that IAP serves as a receptor for TSP-1 in human GC since the recent studies have shown that IAP binds to the C-terminal domain of TSP-1, where no RGD sequence is present (Gao et al., 1996; Dorahy et al., 1997).
TSP-1 is known to interact with extracellular matrices such as fibronectin and collagen (Dardik and Lahav, 1989). This suggests that IAP mediates the interaction between GC and extracellular matrices via TSP-1. Recently, we showed that laminin, a ligand for integrin 6ß1, suppressed progesterone production by human luteinizing GC in vitro and its effect is partially mediated with the interaction between laminin and integrin 6ß1 (Fujiwara et al., 1997), supporting the previous report which proposed the physiological role of extracellular matrices on luteinization process of human GC (Amsterdam et al., 1989). Additionally, we observed that GC produced collagen type IV, a ligand for integrin 2ß1, during luteinization under the stimulation of LH and that collagen type IV suppresses luteinization of GC in an autocrine fashion (Yamada et al., 1999). Similarly, IAP and TSP-1 may serve as local regulators for GC function in concert with integrins and extracellular matrices.
Recent studies have demonstrated that TSP-1 modulates the biological activity of growth factors such as transforming growth factor ß (TGF-ß) and basic fibroblast growth factor (bFGF) (Murphy-Ullrich et al., 1992; Schultz-Cherry et al., 1995; Taraboletti et al., 1997). The binding of TSP-1 to latent TGF-ß produces active TGF-ß. For example, human natural killer cell expansion was reported to be regulated by thrombospondin-mediated activation of TGF-ß1 (Pierson et al., 1996). TGF-ß has been shown to modulate the proliferation and differentiation of rat GC (Feng et al., 1986; Knecht et al., 1986; Dodson and Schomberg, 1987). On the other hand, bFGF have been demonstrated to be involved in folliculogenesis and CL function (Taylor and Clark, 1992; Wordinger et al., 1993). Consequently, it is possible that TSP-1 produced by human GC locally regulates the follicular growth by modulating the activity of TGF-ß and bFGF. TSP-1 is also known to modulate angiogenesis by direct and TGF-ß- and/or bFGF-mediated bidirectional effects (Passaniti et al., 1992; Sheibani and Frazier, 1995; Taraboletti et al., 1997). During CL formation, capillaries from the theca interna penetrate the GC layer and reach the central cavity, and bFGF has been considered to regulate such angiogenesis (Bagavandoss and Wilks, 1991; Zheng et al., 1993). TSP-1 produced by luteinizing GC may participate in the construction of the vascular structure of CL by modulating the angiogenic activity directly or via bFGF. Since TSP-1 is a potent blood coagulation factor (Bornstein, 1992; Mosher et al., 1992), its implication in the process or repair of follicular rupture can be also proposed in the ovulatory phase.
In conclusion, we have demonstrated that IAP is a lineage specific molecule in human granulosa–large luteal cells. Co-expresson of IAP and its ligand, TSP-1, on human GC suggests that TSP-1 has some physiological effect on GC function via IAP in an autocrine fashion. Further investigation of the role of IAP and TSP-1 will contribute to clarifying the physiology of the human ovary.
Acknowledgments
We thank Dr Kazuo Maruyama (The Institute of Medical Science, The University of Tokyo) for providing us pME18S mammalian cell expression plasmid vector. This work was supported in part by Grants-in-Aid for Scientific Research (no. 09671673, 09671674, 09671676).
Notes
4 To whom correspondence should be addressed 
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Submitted on March 18, 1999; accepted on July 12, 1999.
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  J. Greenaway, P. A. Gentry, J.-J. Feige, J. LaMarre, and J. J. Petrik Thrombospondin and Vascular Endothelial Growth Factor Are Cyclically Expressed in an Inverse Pattern During Bovine Ovarian Follicle Development Biol Reprod, May 1, 2005; 72(5): 1071 - 1078. [Abstract] [Full Text] [PDF]
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 T. Higuchi, H. Fujiwara, H. Egawa, Y. Sato, S. Yoshioka, K. Tatsumi, K. Itoh, M. Maeda, J. Fujita, and S. Fujii Cyclic AMP enhances the expression of an extravillous trophoblast marker, melanoma cell adhesion molecule, in choriocarcinoma cell JEG3 and human chorionic villous explant cultures Mol. Hum. Reprod., June 1, 2003; 9(6): 359 - 366. [Abstract] [Full Text] [PDF]
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J. J. Petrik, P. A. Gentry, J.-J. Feige, and J. LaMarre Expression and Localization of Thrombospondin-1 and -2 and Their Cell-Surface Receptor, CD36, During Rat Follicular Development and Formation of the Corpus Luteum Biol Reprod, November 1, 2002; 67(5): 1522 - 1531. [Abstract] [Full Text] [PDF]
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 H. Egawa, H. Fujiwara, T. Hirano, T. Nakayama, T. Higuchi, K. Tatsumi, T. Mori, and S. Fujii Peripheral blood mononuclear cells in early pregnancy promote invasion of human choriocarcinoma cell line, BeWo cells Hum. Reprod., February 1, 2002; 17(2): 473 - 480. [Abstract] [Full Text] [PDF]
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T. Nakayama, H. Fujiwara, M. Maeda, T. Inoue, S. Yoshioka, T. Mori, and S. Fujii Human peripheral blood mononuclear cells (PBMC) in early pregnancy promote embryo invasion in vitro: HCG enhances the effects of PBMC Hum. Reprod., January 1, 2002; 17(1): 207 - 212. [Abstract] [Full Text] [PDF]
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