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Immunohistochemical study of osteopontin and l -selectin in a rat endometriosis model and in human endometriosis
Immunohistochemical study of osteopontin and L-selectin in a rat endometriosis model and in human endometriosis Kohei Odagiri, M.D.,a Ryo Konno, M.D., Ph.D.,a Hiroyuki Fujiwara, M.D., Ph.D.,b Sachiho Netsu, M.D.,a Michitaka Ohwada, M.D., Ph.D.,b Hiroaki Shibahara, M.D., Ph.D.,b and Mitsuaki Suzuki, M.D., Ph.D.b a Department of Gynecology, Omiya Medical Center, Jichi Medical School, Saitama; and b Department of Obstetrics and Gynecology, Jichi Medical School, Shimotsuke City, Japan
Objective: To investigate the localization of the proteins osteopontin (OPN) and L-selectin (SELL). Design: Retrospective nonrandomized immunohistochemical study in a surgically induced rat model of peritoneal endometriosis and in samples of human endometriotic lesions of ovaries. Setting: Department of gynecology in a university hospital. Patient(s): A rat endometriosis model was induced in 10 8-week-old SLC-Sprague-Dawley rats by surgical autotransplantation of uterus. Fourteen premenopausal women with histologically diagnosed endometriosis were selected (mean age, 39 y; range, 25–53 y). Twenty endometriotic specimens were obtained from 14 patients who underwent laparoscopic surgery for enlarged endometriotic cysts. Intervention(s): Histopathological examination of endometriotic ovarian specimens for OPN and SELL expression by immunohistochemistry. Main Outcome Measure(s): Demonstration of the immunoreactive staining of OPN and SELL expressions in tissues of a rat endometriosis model and of human endometriosis. Result(s): In both tissues from a rat endometriosis model and from human endometriosis, OPN was stained more prominently in glandular epithelium than in interstitial space, whereas SELL stained more prominently in interstitial space (macrophages and lymphocytes) than in epithelium. The staining pattern of OPN in ectopic endometriotic lesions was very similar to that in eutopic normal human endometrium in the secretory phase. Conclusion(s): These results suggested important roles for OPN and SELL in the pathogenesis of endometriosis. (Fertil Steril 2007;88(Suppl 2):1207–11. 2007 by American Society for Reproductive Medicine.) Key Words: Endometriosis, osteopontin, l-selectin, tissue remodeling
The pathogenesis of endometriosis is still poorly defined, although many hypotheses have been proposed (1, 2). In our study published elsewhere, we induced a rat endometriosis model and subjected it to transcriptomics to examine the molecules that are related to endometriosis (unpublished data). Transcriptomics demonstrated up-regulation of the expression of 74 genes. Among them, we focused on two molecules, osteopontin (OPN) and L-selectin (SELL), because these molecules are related to cell adhesion and inflammation, which may be involved in endometriosis. Osteopontin is a 70-kDa secreted glycosylated phosphoprotein that was originally isolated from bone matrix (3) and has been found in a wide variety of tissues or substances, including milk, urine, kidney, secretory glands, and some tumor tissues (4, 5). It is secreted not only from luminal
Received October 13, 2005; revised and accepted January 3, 2007. Reprint requests: Ryo Konno, M.D., Ph.D., Department of Gynecology, Omiya Medical Center, Jichi Medical School, Amanuma-cho, Omiya, Saitama, 330-8503, Japan (FAX: 81-48-648-5188; E-mail: kryo77@ excite.co.jp).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2007.02.007
epithelium but also from interstitial cells such as macrophages and lymphocytes. Osteopontin binds especially to avb3, a kind of integrin, and relates to cell adhesion and migration (6, 7). In the field of gynecology, OPN is expressed in endometrium in the mid and late secretory phases (8). Lessey (9) reported that avb3 expression is reduced in endometrium from women with endometriosis, whereas OPN expression is unaffected. But there are no data on the relation between endometriotic tissue and OPN. L-Selectin is a 65- to 75-kDa cell adhesion glycoprotein that is a cell surface component and was originally isolated from lymphocytes (10). This molecule plays an important role in lymphocyte adhesion to vascular endothelium at the sites of inflammation, which is immunologically called the rolling phenomenon, and then enables lymphocytes to migrate from the blood stream to the interstitial space (11). In the field of gynecology, SELL is related to trophoblast adhesion to the uterine wall, which is the requisite first step of implantation and placentation (12). Kao et al. (13) reported that N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of SELL ligands) was down-regulated in
Fertility and Sterility Vol. 88, Suppl 2, October 2007 Copyright ª2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
1207
endometrium from women with endometriosis, but there are no data on the relation between endometriotic tissue and SELL, just as with OPN. We conducted an immunohistochemical study to examine the localization of OPN and SELL on tissues of a rat endometriosis model and of human endometriosis. MATERIALS AND METHODS Rat Endometriosis Model For the induction of an endometriosis model, 10 SLCSprague-Dawley rats (8 wk old) were maintained on a 12:12 hour light–dark schedule for 1 week. Details have been described elsewhere (14) of the surgical technique of autotransplanting 5 mm 5 mm of uterine tissue. We used the same technique with minor modifications (15). Uterine tissue was autotransplanted to bilateral peritoneum. Five rats were chosen as an endometriosis model, and the other five rats were chosen as a control model without autotransplanting of uterine tissue. The induced endometrioses were obtained from five anesthetized endometriosismodel rats, 7 days after uterine autotransplantation. Eutopic endometrium was obtained from five anesthetized control model rats. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After resection, all samples were fixed in 10% buffered formalin and embedded in paraffin for immunohistological examination. Tissue Samples of Human Endometriosis Twenty endometriotic lesions were obtained from the premenopausal Japanese women (mean age, 39 y; range, 25– 53 y), who underwent laparoscopic cystectomy for enlarged chocolate cysts (n ¼ 20). Collection of human materials for this study was approved by the Committee for the Protection of Human Subjects at the Jichi Medical School. Samples from five patients who underwent hysterectomy as a result of uterine cervical cancer with carcinoma in situ were chosen as a control section of human eutopic endometrium. Three of them were in proliferative phase, and the other two specimens were in secretary phase. Operative findings confirmed no endometriosis in a control group. Immunohistochemistry Immunohistochemistry for OPN and SELL was performed on a rat endometriosis model and on tissues of human endometriosis to study the localization of these proteins. Eutopic human endometrial tissues without endometriosis in the proliferative and secretory phase were used as control tissues. The anti-OPN antibody (1B20; American Research Products, Inc., Belmont, MA) is a monoclonal mouse IgG1 antibody that has been purified (protein A chromatography) from hybridoma supernatant against the synthetic peptide of the Cterminus of OPN of human origin. The anti-SELL antibody (lam1-116; Santa Cruz Biotechnology, Inc., Santa Cruz, 1208
Odagiri et al.
CA) is a monoclonal mouse IgG2a antibody that is raised against the lectin domain of SELL of mouse origin. Paraffin-embedded sections were deparaffinized in xylene and rehydrated in ethanol. The antigen retrieval method was performed only for the SELL antibody, by microwaving in citrate buffer (0.1 M citrate acid and 0.1 M natrium citrate, pH 6.0; microwave, 500 W for 5 min). Endogenous peroxidase activity was quenched with 3% hydrogen peroxidase for 5 minutes. Each primary antibody was serially diluted in a solution of phosphate-buffered saline containing 1% bovine serum albumin and 0.02% sodium azide. Tissue sections were incubated with primary antibody for 1 hour at room temperature at the following dilutions: 1B20, 1:5,000, and lam1-116, 1:100. Negative control sections were treated without primary antibodies but with nonimmune serum and secondary antibody. Subsequently, sections were washed with phosphate-buffered saline and incubated with secondary antibody (anti-mouse and anti-rabbit: Dako, Kyoto, Japan) for 45 minutes at room temperature. After rinsing with phosphate-buffered saline, the immunoreactive antigen was visualized by using liquid 3,30 -diaminobenzidine as the chromogen (Dako). Slides were counterstained by using hematoxylin, followed by dehydration in a graded series of ethanols and clearing in xylene. All slides were evaluated by experienced gynecologists (H.F., S.N.) who were blind to the study manipulations. The evaluation of the staining intensities was divided into three ranges: (<5%), þ (550%), and þþ (>50%). Table 1 shows the list of the samples in the study. RESULTS Pathologic Features of Autotransplanted Peritoneal Lesions of Rat Endometriosis Model Seven days after uterine autotransplantation, hematoxylin staining showed that the uterine implants had grown into ellipsoidal cystic structures that were composed of endometriotic cysts, stroma, and fibrotic interstitium, with some inflammatory cells. Endometriotic cysts were detected in all peritoneal lesions (n ¼ 5) in the rat endometriosis model. Fibrotic change also was detected surrounding these cysts. Pathologic Features of Human Endometriotic Tissue Hematoxylin staining showed endometriotic cysts with epithelial cells and stroma. In the interstitial area around endometriotic cysts, fibrosis and smooth muscle metaplasia were detected with some inflammatory cells, including lymphocytes and macrophages. Immunostaining Pattern of OPN No immunostaining was detected in either epithelial or interstitial cells in rat eutopic endometrium. In the examination of a rat endometriosis model, intense immunostaining was
Osteopontin and L-selectin in endometriosis
Vol. 88, Suppl 2, October 2007
TABLE 1 Results of samples used in study with immunohistochemical staining. Staining intensities OPN
SELL
Samples
n
L
D
DD
L
D
DD
Human proliferative endometrium Human secretory endometrium Human endometriosis Rat endometrium Rat endometriosis
3 2 20 5 5
0 0 2 5 1
3 0 2 0 3
0 2 16 0 1
2 2 8 5 2
1 0 8 0 3
0 0 4 0 0
Note: The evaluation of the staining intensity was divided into three categories: (<5%), þ (550%), and þþ (>50%). Odagiri. Osteopontin and L-selectin in endometriosis. Fertil Steril 2007.
detected in epithelial cells of endometriotic cysts. Weak or moderate immunostaining was also detected in some fibroblastic cells in the interstitial area of rat endometriotic lesion. In human eutopic endometrium from control sections, a diffuse staining pattern was detected in epithelial cells of
the functional layer, especially in the secretory phase, whereas a scattered staining pattern was detected in cells of the functional and basal layer in the proliferative phase. Intense OPN expression was present in both the cytoplasm and nucleus of these positive cells. No stromal cells were stained in either the functional or basal layer (Fig. 1).
FIGURE 1 Immunohistochemistry of OPN in eutopic normal human endometrium in the late-secretory phase (A: original magnification, 100; B: original magnification, 400) and human endometriotic tissue (C: original magnification, 100; D: original magnification, 400). The staining pattern of glandular epithelial cells in human endometriotic tissue is diffuse and similar to that of the functional layer in eutopic normal human endometrium in the secretory phase.
Odagiri. Osteopontin and L-selectin in endometriosis. Fertil Steril 2007.
Fertility and Sterility
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With regard to human endometriosis, the staining pattern of epithelial cells of endometriotic cysts was diffuse, which is similar to the staining pattern of eutopic endometrial epithelial cells of the functional layer in the secretory phase in control sections. No immunostaining was detected in interstitial cells of endometriotic lesions (Fig. 1). Immunostaining Pattern of SELL No immunostaining was detected in rat eutopic endometrium. With regard to the rat endometriosis model, some inflammatory cells in interstitial areas surrounding endometriotic cysts were weakly stained, whereas none of the epithelial cells of endometriotic cysts was stained. No immunostaining was detected in either epithelial or stromal cells in eutopic human endometrium of control sections. In human endometriosis, intense immunostaining was detected in interstitial cells, including lymphocytes and macrophages, whereas no immunostaining was detected in epithelium (Fig. 2). DISCUSSION In this study, we demonstrated the intense expression of OPN in eutopic normal human endometrium. Its localization was
especially positive in the functional layer in the secretary phase, and its staining pattern was diffuse. In both tissues from the rat endometriosis model and human endometriosis, the staining pattern for OPN was significantly more intense in the epithelial cells of endometriotic cysts than in the interstitial space, whereas the staining pattern for SELL had a diverse distribution. The morphological pattern of endometriotic tissue was similar to that of eutopic endometrium in the proliferative phase. However, diffuse and intense expression for OPN was similar between epithelial cells in eutopic endometrium in the secretory phase and in endometriotic cyst. To our knowledge, there are no data on the relationship between OPN, SELL, and endometriosis, although there have been some studies on the relationship between OPN, SELL, and eutopic human endometrium (8). So far, there are two major theories in the literature for the pathogenesis of endometriosis: implantation theory and metaplasia theory (1, 2). In our study, we focused on the similarity of the staining pattern for OPN between epithelial cells in normal human endometrium and in human endometriosis. Osteopontin is a secreted protein that is related to cell adhesion and migration. Therefore, we speculate in accordance with implantation theory (1) that endometriotic cells with
FIGURE 2 Immunohistochemistry of SELL in eutopic normal human endometrium in the secretory phase (D: original magnification, 200) and in human endometriotic tissue (A: original magnification, 100, B and C: original magnification, 400). Eutopic endometrium shows no immunostaining. In endometriotic tissues, interstitial cells are intensively stained (arrows), whereas no epithelial cells are stained.
Odagiri. Osteopontin and L-selectin in endometriosis. Fertil Steril 2007.
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Osteopontin and L-selectin in endometriosis
Vol. 88, Suppl 2, October 2007
intense expression for OPN originally flow in a retrograde manner from epithelial cells in the eutopic endometrium in the secretory phase, followed by the menstrual period, and are implanted to ectopic lesions. Furthermore, in our study, SELL was intensely expressed in interstitial lesion in endometriotic lesion. L-Selectin is an adhesion protein that is expressed on the surface of T lymphocytes to bind to vascular endothelium via sialomucin and migrate to the interstitial space at the site of inflammation. Many lymphocytes can be seen in endometriotic lesions. These lymphocytes may be derived from the vascular space and produce many cytokines that are related to inflammation and progression of endometriosis. Therefore, we speculate in accordance with metaplasia theory (2) that endometriotic cells are affected by many cytokines that cause metaplastic change to form an endometriotic lesion. Of course, we do not believe that we could prove these speculations completely by only our result. But it is clear that OPN and SELL may have some roles in the course of the progression of endometriosis. We believe that endometriosis is based on both theories, implantation and metaplasia, not on only one theory. The development of endometriosis is not under the control of only a single mechanism; rather, a network of many cytokines and microenvironmental factors is related to this disease. Further studies are necessary to determine the validity of these speculations to clarify the pathogenesis of endometriosis. Acknowledgment: The authors are grateful to Masao Sugamata, M.D., Ph.D., for valuable discussions and helpful technical assistance.
REFERENCES 1. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the pelvic cavity. Am J Obstet Gynecol 1927;14: 422–69.
Fertility and Sterility
2. Matsuura K, Ohtake H, Katabuchi H, Okamura H. Coelomic metaplasia theory of endometriosis: evidence from in vivo studies and an in vitro experimental model. Gynecol Obstet Invest 1999;47:18–22. 3. Reinholt FP, Hultenby K, Oldberg A, Heinegard D. Osteopontin—a possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA 1990;87:4473–5. 4. Brown LF, Berse B, Van de Water L, Papadopoulos-Sergiou A, Perruzzi CA, Manseau EJ, et al. Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surface. Mol Biol Cell 1992;3:1169–80. 5. Coppola D, Szabo M, Boulware D, Muraca P, Alsarraj M, Chambers AF, et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res 2004;10:184–90. 6. Hu DD, Lin EC, Kovach NL, Hoyer JR, Smith JW. A biochemical characterization of the binding of osteopontin to integrins avb1 and avb5. J Biol Chem 1995;270:26232–8. 7. Liaw L, Skinner MP, Raines EW, Ross R, Cheresh DA, Schwartz SM, et al. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins: role of avb3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest 1995;95:713–24. 8. Apparao KB, Murray MJ, Fritz MA, Meyer WR, Chambers AF, Truong PR, et al. Osteopontin and its receptor avb3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol Metab 2001;86:4991–5000. 9. Lessey BA. Implantation defects in infertile women with endometriosis. Ann NY Acad Sci 2002;955:265–80. 10. Tu L, Poe JC, Kadono T, Venturi GM, Bullard DC, Tedder TF, et al. A functional role for circulating mouse L-selectin in regulating leukocyte/endothelial cell interactions in vivo. J Immunol 2002;169:2034–43. 11. Biancone L, Cantaluppi V, Duo D, Deregibus MC, Torre C, Camussi G, et al. Role of L-selectin in the vascular homing of peripheral bloodderived endothelial progenitor cells. J Immunol 2004;173:5268–74. 12. Genbacev OD, Prakobphol A, Foulk RA, Krtolica AR, Ilic D, Singer MS, et al. Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface. Science 2003;299:405–8. 13. Kao LC, Germeyer A, Tulac S, Lobo S, Yang JP, Taylor RN, et al. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology 2003;144:2870–81. 14. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684–94. 15. Uchiide I, Ihara T, Sugamata M. Pathological evaluation of the rat endometriosis model. Fertil Steril 2002;78:782–6.
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Objective: To investigate the localization of the proteins osteopontin (OPN) and L-selectin (SELL). Design: Retrospective nonrandomized immunohistochemical study in a surgically induced rat model of peritoneal endometriosis and in samples of human endometriotic lesions of ovaries. Setting: Department of gynecology in a university hospital. Patient(s): A rat endometriosis model was induced in 10 8-week-old SLC-Sprague-Dawley rats by surgical autotransplantation of uterus. Fourteen premenopausal women with histologically diagnosed endometriosis were selected (mean age, 39 y; range, 25–53 y). Twenty endometriotic specimens were obtained from 14 patients who underwent laparoscopic surgery for enlarged endometriotic cysts. Intervention(s): Histopathological examination of endometriotic ovarian specimens for OPN and SELL expression by immunohistochemistry. Main Outcome Measure(s): Demonstration of the immunoreactive staining of OPN and SELL expressions in tissues of a rat endometriosis model and of human endometriosis. Result(s): In both tissues from a rat endometriosis model and from human endometriosis, OPN was stained more prominently in glandular epithelium than in interstitial space, whereas SELL stained more prominently in interstitial space (macrophages and lymphocytes) than in epithelium. The staining pattern of OPN in ectopic endometriotic lesions was very similar to that in eutopic normal human endometrium in the secretory phase. Conclusion(s): These results suggested important roles for OPN and SELL in the pathogenesis of endometriosis. (Fertil Steril 2007;88(Suppl 2):1207–11. 2007 by American Society for Reproductive Medicine.) Key Words: Endometriosis, osteopontin, l-selectin, tissue remodeling
The pathogenesis of endometriosis is still poorly defined, although many hypotheses have been proposed (1, 2). In our study published elsewhere, we induced a rat endometriosis model and subjected it to transcriptomics to examine the molecules that are related to endometriosis (unpublished data). Transcriptomics demonstrated up-regulation of the expression of 74 genes. Among them, we focused on two molecules, osteopontin (OPN) and L-selectin (SELL), because these molecules are related to cell adhesion and inflammation, which may be involved in endometriosis. Osteopontin is a 70-kDa secreted glycosylated phosphoprotein that was originally isolated from bone matrix (3) and has been found in a wide variety of tissues or substances, including milk, urine, kidney, secretory glands, and some tumor tissues (4, 5). It is secreted not only from luminal
Received October 13, 2005; revised and accepted January 3, 2007. Reprint requests: Ryo Konno, M.D., Ph.D., Department of Gynecology, Omiya Medical Center, Jichi Medical School, Amanuma-cho, Omiya, Saitama, 330-8503, Japan (FAX: 81-48-648-5188; E-mail: kryo77@ excite.co.jp).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2007.02.007
epithelium but also from interstitial cells such as macrophages and lymphocytes. Osteopontin binds especially to avb3, a kind of integrin, and relates to cell adhesion and migration (6, 7). In the field of gynecology, OPN is expressed in endometrium in the mid and late secretory phases (8). Lessey (9) reported that avb3 expression is reduced in endometrium from women with endometriosis, whereas OPN expression is unaffected. But there are no data on the relation between endometriotic tissue and OPN. L-Selectin is a 65- to 75-kDa cell adhesion glycoprotein that is a cell surface component and was originally isolated from lymphocytes (10). This molecule plays an important role in lymphocyte adhesion to vascular endothelium at the sites of inflammation, which is immunologically called the rolling phenomenon, and then enables lymphocytes to migrate from the blood stream to the interstitial space (11). In the field of gynecology, SELL is related to trophoblast adhesion to the uterine wall, which is the requisite first step of implantation and placentation (12). Kao et al. (13) reported that N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of SELL ligands) was down-regulated in
Fertility and Sterility Vol. 88, Suppl 2, October 2007 Copyright ª2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
1207
endometrium from women with endometriosis, but there are no data on the relation between endometriotic tissue and SELL, just as with OPN. We conducted an immunohistochemical study to examine the localization of OPN and SELL on tissues of a rat endometriosis model and of human endometriosis. MATERIALS AND METHODS Rat Endometriosis Model For the induction of an endometriosis model, 10 SLCSprague-Dawley rats (8 wk old) were maintained on a 12:12 hour light–dark schedule for 1 week. Details have been described elsewhere (14) of the surgical technique of autotransplanting 5 mm 5 mm of uterine tissue. We used the same technique with minor modifications (15). Uterine tissue was autotransplanted to bilateral peritoneum. Five rats were chosen as an endometriosis model, and the other five rats were chosen as a control model without autotransplanting of uterine tissue. The induced endometrioses were obtained from five anesthetized endometriosismodel rats, 7 days after uterine autotransplantation. Eutopic endometrium was obtained from five anesthetized control model rats. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After resection, all samples were fixed in 10% buffered formalin and embedded in paraffin for immunohistological examination. Tissue Samples of Human Endometriosis Twenty endometriotic lesions were obtained from the premenopausal Japanese women (mean age, 39 y; range, 25– 53 y), who underwent laparoscopic cystectomy for enlarged chocolate cysts (n ¼ 20). Collection of human materials for this study was approved by the Committee for the Protection of Human Subjects at the Jichi Medical School. Samples from five patients who underwent hysterectomy as a result of uterine cervical cancer with carcinoma in situ were chosen as a control section of human eutopic endometrium. Three of them were in proliferative phase, and the other two specimens were in secretary phase. Operative findings confirmed no endometriosis in a control group. Immunohistochemistry Immunohistochemistry for OPN and SELL was performed on a rat endometriosis model and on tissues of human endometriosis to study the localization of these proteins. Eutopic human endometrial tissues without endometriosis in the proliferative and secretory phase were used as control tissues. The anti-OPN antibody (1B20; American Research Products, Inc., Belmont, MA) is a monoclonal mouse IgG1 antibody that has been purified (protein A chromatography) from hybridoma supernatant against the synthetic peptide of the Cterminus of OPN of human origin. The anti-SELL antibody (lam1-116; Santa Cruz Biotechnology, Inc., Santa Cruz, 1208
Odagiri et al.
CA) is a monoclonal mouse IgG2a antibody that is raised against the lectin domain of SELL of mouse origin. Paraffin-embedded sections were deparaffinized in xylene and rehydrated in ethanol. The antigen retrieval method was performed only for the SELL antibody, by microwaving in citrate buffer (0.1 M citrate acid and 0.1 M natrium citrate, pH 6.0; microwave, 500 W for 5 min). Endogenous peroxidase activity was quenched with 3% hydrogen peroxidase for 5 minutes. Each primary antibody was serially diluted in a solution of phosphate-buffered saline containing 1% bovine serum albumin and 0.02% sodium azide. Tissue sections were incubated with primary antibody for 1 hour at room temperature at the following dilutions: 1B20, 1:5,000, and lam1-116, 1:100. Negative control sections were treated without primary antibodies but with nonimmune serum and secondary antibody. Subsequently, sections were washed with phosphate-buffered saline and incubated with secondary antibody (anti-mouse and anti-rabbit: Dako, Kyoto, Japan) for 45 minutes at room temperature. After rinsing with phosphate-buffered saline, the immunoreactive antigen was visualized by using liquid 3,30 -diaminobenzidine as the chromogen (Dako). Slides were counterstained by using hematoxylin, followed by dehydration in a graded series of ethanols and clearing in xylene. All slides were evaluated by experienced gynecologists (H.F., S.N.) who were blind to the study manipulations. The evaluation of the staining intensities was divided into three ranges: (<5%), þ (550%), and þþ (>50%). Table 1 shows the list of the samples in the study. RESULTS Pathologic Features of Autotransplanted Peritoneal Lesions of Rat Endometriosis Model Seven days after uterine autotransplantation, hematoxylin staining showed that the uterine implants had grown into ellipsoidal cystic structures that were composed of endometriotic cysts, stroma, and fibrotic interstitium, with some inflammatory cells. Endometriotic cysts were detected in all peritoneal lesions (n ¼ 5) in the rat endometriosis model. Fibrotic change also was detected surrounding these cysts. Pathologic Features of Human Endometriotic Tissue Hematoxylin staining showed endometriotic cysts with epithelial cells and stroma. In the interstitial area around endometriotic cysts, fibrosis and smooth muscle metaplasia were detected with some inflammatory cells, including lymphocytes and macrophages. Immunostaining Pattern of OPN No immunostaining was detected in either epithelial or interstitial cells in rat eutopic endometrium. In the examination of a rat endometriosis model, intense immunostaining was
Osteopontin and L-selectin in endometriosis
Vol. 88, Suppl 2, October 2007
TABLE 1 Results of samples used in study with immunohistochemical staining. Staining intensities OPN
SELL
Samples
n
L
D
DD
L
D
DD
Human proliferative endometrium Human secretory endometrium Human endometriosis Rat endometrium Rat endometriosis
3 2 20 5 5
0 0 2 5 1
3 0 2 0 3
0 2 16 0 1
2 2 8 5 2
1 0 8 0 3
0 0 4 0 0
Note: The evaluation of the staining intensity was divided into three categories: (<5%), þ (550%), and þþ (>50%). Odagiri. Osteopontin and L-selectin in endometriosis. Fertil Steril 2007.
detected in epithelial cells of endometriotic cysts. Weak or moderate immunostaining was also detected in some fibroblastic cells in the interstitial area of rat endometriotic lesion. In human eutopic endometrium from control sections, a diffuse staining pattern was detected in epithelial cells of
the functional layer, especially in the secretory phase, whereas a scattered staining pattern was detected in cells of the functional and basal layer in the proliferative phase. Intense OPN expression was present in both the cytoplasm and nucleus of these positive cells. No stromal cells were stained in either the functional or basal layer (Fig. 1).
FIGURE 1 Immunohistochemistry of OPN in eutopic normal human endometrium in the late-secretory phase (A: original magnification, 100; B: original magnification, 400) and human endometriotic tissue (C: original magnification, 100; D: original magnification, 400). The staining pattern of glandular epithelial cells in human endometriotic tissue is diffuse and similar to that of the functional layer in eutopic normal human endometrium in the secretory phase.
Odagiri. Osteopontin and L-selectin in endometriosis. Fertil Steril 2007.
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1209
With regard to human endometriosis, the staining pattern of epithelial cells of endometriotic cysts was diffuse, which is similar to the staining pattern of eutopic endometrial epithelial cells of the functional layer in the secretory phase in control sections. No immunostaining was detected in interstitial cells of endometriotic lesions (Fig. 1). Immunostaining Pattern of SELL No immunostaining was detected in rat eutopic endometrium. With regard to the rat endometriosis model, some inflammatory cells in interstitial areas surrounding endometriotic cysts were weakly stained, whereas none of the epithelial cells of endometriotic cysts was stained. No immunostaining was detected in either epithelial or stromal cells in eutopic human endometrium of control sections. In human endometriosis, intense immunostaining was detected in interstitial cells, including lymphocytes and macrophages, whereas no immunostaining was detected in epithelium (Fig. 2). DISCUSSION In this study, we demonstrated the intense expression of OPN in eutopic normal human endometrium. Its localization was
especially positive in the functional layer in the secretary phase, and its staining pattern was diffuse. In both tissues from the rat endometriosis model and human endometriosis, the staining pattern for OPN was significantly more intense in the epithelial cells of endometriotic cysts than in the interstitial space, whereas the staining pattern for SELL had a diverse distribution. The morphological pattern of endometriotic tissue was similar to that of eutopic endometrium in the proliferative phase. However, diffuse and intense expression for OPN was similar between epithelial cells in eutopic endometrium in the secretory phase and in endometriotic cyst. To our knowledge, there are no data on the relationship between OPN, SELL, and endometriosis, although there have been some studies on the relationship between OPN, SELL, and eutopic human endometrium (8). So far, there are two major theories in the literature for the pathogenesis of endometriosis: implantation theory and metaplasia theory (1, 2). In our study, we focused on the similarity of the staining pattern for OPN between epithelial cells in normal human endometrium and in human endometriosis. Osteopontin is a secreted protein that is related to cell adhesion and migration. Therefore, we speculate in accordance with implantation theory (1) that endometriotic cells with
FIGURE 2 Immunohistochemistry of SELL in eutopic normal human endometrium in the secretory phase (D: original magnification, 200) and in human endometriotic tissue (A: original magnification, 100, B and C: original magnification, 400). Eutopic endometrium shows no immunostaining. In endometriotic tissues, interstitial cells are intensively stained (arrows), whereas no epithelial cells are stained.
Odagiri. Osteopontin and L-selectin in endometriosis. Fertil Steril 2007.
1210
Odagiri et al.
Osteopontin and L-selectin in endometriosis
Vol. 88, Suppl 2, October 2007
intense expression for OPN originally flow in a retrograde manner from epithelial cells in the eutopic endometrium in the secretory phase, followed by the menstrual period, and are implanted to ectopic lesions. Furthermore, in our study, SELL was intensely expressed in interstitial lesion in endometriotic lesion. L-Selectin is an adhesion protein that is expressed on the surface of T lymphocytes to bind to vascular endothelium via sialomucin and migrate to the interstitial space at the site of inflammation. Many lymphocytes can be seen in endometriotic lesions. These lymphocytes may be derived from the vascular space and produce many cytokines that are related to inflammation and progression of endometriosis. Therefore, we speculate in accordance with metaplasia theory (2) that endometriotic cells are affected by many cytokines that cause metaplastic change to form an endometriotic lesion. Of course, we do not believe that we could prove these speculations completely by only our result. But it is clear that OPN and SELL may have some roles in the course of the progression of endometriosis. We believe that endometriosis is based on both theories, implantation and metaplasia, not on only one theory. The development of endometriosis is not under the control of only a single mechanism; rather, a network of many cytokines and microenvironmental factors is related to this disease. Further studies are necessary to determine the validity of these speculations to clarify the pathogenesis of endometriosis. Acknowledgment: The authors are grateful to Masao Sugamata, M.D., Ph.D., for valuable discussions and helpful technical assistance.
REFERENCES 1. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the pelvic cavity. Am J Obstet Gynecol 1927;14: 422–69.
Fertility and Sterility
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