Department of Obstetrics and Gynecology, University of Missouri, Columbia, MO, 65212, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: endometriosis/endometriosis protein-I/endometrium/haptoglobin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our prior research has demonstrated that in vitro, both rat and human endometriotic lesions synthesize and secrete a protein which we called endometriosis protein-I (ENDO-I; Sharpe and Vernon, 1993; Sharpe et al., 1993). We have subsequently shown that partial ENDO-I amino acid sequence and full-length ENDO-I cDNA sequence are virtually identical to those of hepatic haptoglobin (Sharpe-Timms et al., 1998; Piva and Sharpe-Timms, 1999
). As recent data suggest that hepatic haptoglobin possesses novel immunosuppressive and angiogenic activities in association with certain disease states (for a review, see Dobryszycka, 1997), expression and localization of extrahepatic haptoglobin (ENDO-I) in the peritoneal cavity may contribute to the pathogenesis of endometriosis by altering immune function or promoting neovascularization of developing endometriotic lesions. Therefore, this study evaluated in-vivo expression and localization of ENDO-I in human endometrial and endometriotic tissues.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In-situ hybridization
Site-specific ENDO-I mRNA expression was identified in tissue sections by non-isotopic in-situ hybridization using digoxin (Dig)-labelled antisense and sense riboprobes generated from cDNA as previously described in detail and validated (Olson et al., 1997). The riboprobes used in these experiments were originally developed to study the extrahepatic haptoglobin called glycoprotein-42 (GP-42), a gene expressed by rabbit endometrium at the time of embryo implantation (Hoffman et al., 1996
; Olson et al., 1997
). Briefly, the haptoglobin 5' cDNA probes were prepared by polymerase chain reaction (PCR) amplification. The 5' PCR primer design was based on a highly conserved region of the ß-chain N-terminal amino acid sequences, especially between residues 7 and 13, contained in GP-42 (Olson et al., 1997
), rabbit hepatic haptoglobin (Chow et al., 1983
) and human hepatic haptoglobin (Maeda, 1985
). This region was also conserved in the ENDO-I ß-chain N-terminal amino acid sequence (Sharpe-Timms et al., 1998
). The program Primer Designer (Scientific and Educational Software, Durham, NC, USA) and the human hepatic haptoglobin ß-chain nucleotide sequences (Maeda, 1985
) were used to find a compatible 3' primer (Olson et al., 1997
). The sequence for the 3' primer was also confirmed within the human ENDO-I nucleotide sequence (Piva and Sharpe-Timms, 1999
).
In contrast to previous studies (Olson et al., 1997) in which frozen tissue sections were prepared, formalin-fixed, paraffin-embedded tissues were used in this study. Tissues were sectioned at 5 µm and dried on glass slides at 60°C for 50min. Tissue sections were de-waxed in two changes of fresh xylene for 5 min each, re-hydrated through a series of ethanols (100%, 100%, 95%, 70%) for 1min each, and washed twice in two changes of diethylpyrocarbonate (DEPC-treated water. In-situ hybridization using the Dig-labelled antisense (positive detection) and sense (negative control) riboprobes (diluted 1:800) and subsequent immunological detection (1:500 dilution of alkaline phosphatase-conjugated anti-Dig antibody) was then performed as described previously (Olson et al., 1997
). Endometrial and endometriotic glandular epithelium also served as an internal negative control for this procedure, as we have previously shown that isolated endometrial glands do not synthesize ENDO-I de novo (Sharpe et al., 1993
).
Immunohistochemistry
Immunohistochemistry was used to identify site-specific ENDO-I protein localization. Semi-adjacent sections (5 µm) of the formalin-fixed, paraffin-embedded tissues used for the in-situ hybridization were prepared for immunohistochemistry. Immunohistochemistry was performed using an avidinbiotin complex peroxidase procedure according to manufacturer's instructions (Vectastain ABC, Vector, Burlingame, CA, USA) and as used previously in our laboratory to study immunolocalization of granulocyte-macrophage colony-stimulating factor in similar tissues (Sharpe-Timms et al., 1994). A polyclonal anti-haptoglobin antibody (Dako, CA, USA) which we have shown previously to recognize ENDO-I protein (Sharpe-Timms et al., 1998
), was used as the primary antibody for the ENDO-I immunohistochemical staining. Tissue sections were also incubated with normal rabbit sera or phosphate-buffered saline solution substituted for primary antibody as negative controls for the immunohistochemical staining (not shown; absence of staining corresponded to endometrial glands which served as internal negative controls). Peroxidase activity was demonstrated by incubation with 3,3'- diaminobenzidine substrate, yielding a brown intracellular precipitate; this confirmed peroxidase staining. Sections were counterstained with haematoxylin.
Data analysis
Expression and localization differences were evaluated and scored on an inverted phase-contrast microscope by two different observers blinded to the tissue origin using the H-SCORE system (Lessey et al., 1994). The H-SCORE, representing levels of staining intensity and distribution, is calculated using the following equation:
![]() |
where I is the intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong respectively), and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%. Intra-observer variation was <5%, and inter-observer variation was <10%; data were averaged between observers. Photomicrographs were made using Kodak 400 ASA film (Eastman Kodak, Rochester, NY, USA).
In-situ hybridization and immunohistochemical H-SCORE data passed both normality testing (P = 0.068, P = 0.511 respectively) and equal variance testing (P = 0.247, P = 0.103 respectively) as determined using SigmaStat Statistical Software (Jandel Scientific Software, San Rafael, CA, USA). Differences in the mean level of ENDO-I mRNA expression between tissues (endometrium from controls, endometrium from women with endometriosis, and endometriotic lesions) during the stages of the menstrual cycle (proliferative and secretory cycle stages) were evaluated using two-way analysis of variance (ANOVA) using SigmaStat Statistical Software. The two-way ANOVA statistical model used was:
ENDO-I mRNA expression = tissuexcycle stagex(tissuexcycle stage interaction)
When F-testing indicated significance in all variables including tissuexcycle stage interaction, the effects of cycle stage on ENDO-I mRNA expression by the various tissues were evaluated using Tukey's multiple comparison tests.
The same two-way ANOVA model was used to evaluate differences in the mean level of ENDO-I protein localization. As no statistical difference in ENDO-I protein localization was noted for the cycle stage variable (P = 0.142), the data from the proliferative and secretory stages were pooled within each tissue type (endometrium from controls, endometrium from women with endometriosis, and endometriotic lesions) and a one-way ANOVA was performed. Tukey's multiple comparison post-hoc analysis was subsequently used to evaluate determine differences in ENDO-I protein localization between the tissue types.
Differences in the proportions of eutopic endometrial tissues from women with or without endometriosis expressing ENDO-I mRNA or ENDO-I protein were evaluated using Fisher's exact test for two-tailed probability.
Results
Site-specific expression and localization of ENDO-I mRNA and protein
When present, endometrial ENDO-I mRNA was expressed by the functionalis zone of the endometrial stroma, but not the basalis zone (Figure 1A and B). Overall, more (88%) of eutopic endometrial tissues from women with endometriosis expressed ENDO-I mRNA than endometrial tissues from women without endometriosis (50%; P < 0.014). Endometriotic lesion ENDO-I mRNA was expressed by the stroma with preferential localization noted towards peripheral surfaces of the lesions (Figure 2A
D). ENDO-I mRNA was observed in all endometriotic lesions (100%) tested.
|
|
Statistical analysis of differences in amounts of ENDO-I mRNA
Two-way ANOVA of ENDO-I mRNA H-SCORES demonstrated a significant difference in the mean level of ENDO-I mRNA expression among the three types of tissue tested (i.e. endometriotic lesions, eutopic endometrium from women with endometriosis, and eutopic endometrium from women without endometriosis; P = 0.002), between the stages of the menstrual cycle day at which the tissues were collected (proliferative versus secretory; P = 0.032), and a significant statistical interaction between the tissue and stage of the cycle at which it was collected (P = 0.026). Tukey's post-hoc analyses demonstrated that during the secretory stage of the menstrual cycle, endometriotic lesions and eutopic endometrium from women with endometriosis expressed more ENDO-I mRNA than their proliferative phase counterparts, or than either proliferative or secretory eutopic endometrium from women without endometriosis (P < 0.001; Table I).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stimulation of angiogenesis is another newly recognized biological function of haptoglobin. Using an established in-vitro model of angiogenesis, haptoglobin was identified as an angiogenic factor in sera from patients with systemic vasculitis (Cid et al., 1993). Angiogenic activity is elevated in the peritoneal fluid of women with endometriosis (Oosterlynck et al., 1993
; McLaren et al., 1995
, 1996
), yet the key mediators of angiogenesis and their sources in the peritoneal cavity of women with endometriosis are not clear. We postulate that ENDO-I may interact with peritoneal endothelial cells to promote neovascularization of the lesions in women with endometriosis. Indeed, endometriotic implants from rats with surgically induced disease (Vernon and Wilson, 1985
) produce ENDO-I (Sharpe et al., 1993
) and are highly vascularized within 40 days of implantation, with considerable amounts of neovascularization as early as 36 h post-implantation (K.E.Cox and K.L.Sharpe-Timms, unpublished observation). Ongoing investigations in our laboratory concern the angiogenic potential of ENDO-I and its role in neovascularization of endometriotic lesions.
These and other novel functions for haptoglobin have been inferred, in part, through differential glycosylation (van Dijk et al., 1994; Turner, 1995
). Glycans can act as recognition and anti-recognition signals in immune function and glycan receptors (or membrane lectins) function in cellcell recognition, adhesion, cell differentiation and therefore directly affect neighbouring cellular interactions (Montreuil, 1996
; Biermann et al., 1997
; Clark et al., 1997
). Preliminary analysis of ENDO-I carbohydrate composition has revealed the presence of specific glycans (Sharpe-Timms et al., 2000b
; Piva and Sharpe-Timms, 2000
) that could provide a mechanism by which ENDO-I may misdirect local immune response, function in adhesion of endometrial cells in the peritoneal cavity, elicit differentiation of endothelial cells to promote neovascularization of the endometriotic lesions, and may explain whyalthough retrograde menstruation is nearly universalonly some women have implantation of ectopic endometrium in the peritoneal cavity.
In summary, these studies recapitulate our earlier in-vitro investigations showing that endometriotic lesions produce significant amounts of ENDO-I (Sharpe and Vernon, 1993; Sharpe et al., 1993
) and document that endometriotic lesions also produce ENDO-I in vivo (Sharpe-Timms et al., 1998
; Piva and Sharpe-Timms, 1999
). Furthermore, these studies provide novel evidence that eutopic endometrium from women with endometriosis expresses significantly more ENDO-I mRNA as compared with eutopic endometrium from women without this disease during the secretory stage of the menstrual cycle. This difference was not previously detected when reverse-transcription PCR (RT-PCR) was used to evaluate relative ENDO-I expression in similar tissues (Piva and Sharpe-Timms, 1999
), and may be due to differences in methodology. Elevated amounts of ENDO-I protein localization observed in endometriotic lesions and eutopic endometrium from women with endometriosis, regardless of the menstrual cycle stage, may reflect the immunohistochemical detection of hepatic haptoglobin as well as ENDO-I. Endometriotic tissues or dysfunctional endometrial tissues might contain more blood due to increased capillary permeability, and therefore contain more haptoglobin. Yet overall, the fact that more ENDO-I is produced by, and localizes in, eutopic endometrium from women with endometriosis suggests that ENDO-I may be useful in the development of greatly needed, minimally invasive strategies to provide early detection of endometriosis and/or to facilitate the development of unprecedented therapeutic approaches for the management of this enigmatic disease. These developments could significantly reduce the substantial health burden attributed to this malady and increase the reproductive capacity of afflicted women.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berkova, N., Gilbert, C., Goupil, S. et al. (1999) TNF-induced haptoglobin release from human neutrophils: pivotal role of the TNF p55 receptor. J. Immunol., 162, 62266232.
Biermann, L., Gabius, H.-J. and Denker, H.-W. (1997) Neoglycoprotein-binding sites (endogenous lectins) in the fallopian tube, uterus and blastocyst of the rabbit during the preimplantation phase and implantation. Acta Anat., 160, 159171.[ISI][Medline]
Chow, V., Murray, R.K., Dixon, J.D. and Kurosky, A. (1983) Biosynthesis of rabbit haptoglobin: chemical evidence for a single chain precursor. FEBS Lett., 153, 275279.[ISI][Medline]
Cid, M.C., Grant, D.S., Hoffman, G.S. et al. (1993) Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J. Clin. Invest., 91, 977983.[ISI][Medline]
Clark, G.F., Dell, A., Morris, H.R. et al. (1997) Viewing AIDS from a glycobiological perspective: potential linkages to the human feto-embryonic defense system hypothesis. Mol. Hum. Reprod., 3, 513.[Abstract]
Dobryszycka, W. (1997) Biological functions of haptoglobin New pieces to an old puzzle. Eur. J. Clin. Chem. Clin. Biochem., 35, 647654.[ISI][Medline]
Dunselman, G.A.J., Hendrix, M.G.R., Bouckaert, P.X.J.M. and Evers, J.L.H. (1988) Functional aspects of peritoneal macrophages in endometriosis of women. J. Reprod. Fertil., 82, 707710.[Abstract]
Fedele, L., Marchini, M., Bianchi, S. et al. (1990) Structural and ultrastructural defects in preovulatory endometrium of normo-ovulating infertile women with minimal or mild endometriosis. Fertil. Steril., 53, 989993.[ISI][Medline]
Gebel, H.M., Braun, D.P., Tambur, A. et al. (1998) Spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis. Fertil. Steril., 69, 10421047.[ISI][Medline]
Halme, J., Becker, S. and Haskill, S. (1987) Altered maturation and function of peritoneal macrophages: possible role in pathogenesis of endometriosis. Am. J. Obstet. Gynecol., 156, 783789.[ISI][Medline]
Hoffman, L.H., Winfrey, V.P., Blaeuer, G.L. et al. (1996) A haptoglobin-like glycoprotein is produced by implantation-stage rabbit endometrium. Biol. Reprod., 55, 176184.[Abstract]
Isaacson, K.B., Galman, M., Coutifaris, C. et al. (1990) Endometrial synthesis and secretion of complement component-3 by patients with and without endometriosis. Fertil. Steril., 53, 836841.[ISI][Medline]
Ishihara, S., Taketani, T. and Mizuno, M. (1991) Endometriosis impairs glycogen synthesis in human endometrium. Int. J. Fertil., 36, 287290.[ISI][Medline]
Lessey, B.A., Castelbaum, A.J., Sawin, S.W. et al. (1994) Aberrant integrin expression in the endometrium of women with endometriosis. J. Clin. Endocrinol. Metab., 79, 643649.[Abstract]
Maeda, N. (1985) Nucleotide sequence of the haptoglobin and haptoglobin-related gene pair. J. Biol. Chem., 260, 66986709.
McBean, J.H. and Brunsted, J.R. (1993) In vitro CA-125 secretion by endometrium from women with advanced endometriosis. Fertil. Steril., 59, 8992.[ISI][Medline]
McLaren, J., Prentice, A., Charnock-Jones, D.S. et al. (1995) Vascular endothelial growth factor concentrations are elevated in peritoneal fluid in of women with endometriosis. Hum. Reprod., 11, 220223.[Abstract]
McLaren, J., Prentice, A., Charnock-Jones, D.S. et al. (1996) Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J. Clin. Invest., 98, 482489.
Montreuil, J. (1996) Glycobiology: general aspects. In Dumitriu, S. (ed.), Polysaccharides in Medicinal Applications. Marcel Dekker, Inc., New York, pp. 265272.
Noble, L.S., Simpson, E.R., Johns, A. et al. (1996) Aromatase expression in endometriosis. J. Clin. Endocrinol. Metab., 81, 174179.[Abstract]
Noyes, R.W., Hertig, A.F. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 325.[ISI][Medline]
Olson, G.E., Winfrey, V.P., Matrisian, P.E. et al. (1997) Specific expression of haptoglobin mRNA in implantation-stage rabbit uterine epithelium. J. Endocrinol., 152, 6980.[Abstract]
Oosterlynck, D.J., Meuleman, C., Sobis, H. et al. (1993) Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil. Steril., 59, 778782.[ISI][Medline]
Osteen, K.G., Bruner, K.L. and Sharpe-Timms, K.L. (1996) Steroids and growth factor regulation of matrix metalloproteinases expression and the disease endometriosis. Semin. Reprod. Endocrinol., 14, 247255.[ISI][Medline]
Piva, M. and Sharpe-Timms, K.L. (1999) Peritoneal endometriotic lesions differentially expresses a haptoglobin-like gene. Mol. Hum. Reprod., 5, 7178.
Piva, M. and Sharpe-Timms, K.L. (2000) Differential glycosylation and altered lectin binding of endometrial recombinant endometriosis protein-I vs. hepatic haptoglobin [Abstract P-641]. In Proceedings of the Society of Gynecological Investestigations, Chicago, IL.
Piva, M., Knitting, R.A., Griffin, W.T. et al. (1998) Interleukin-6 (IL-6) up regulates expression of Endometriosis Protein-I (ENDO-I) mRNA, a haptoglobin-like gene that is differentially expressed by pelvic endometriosis [Abstract O-171]. In Proceedings of the American Society of Reproductive Medicine, San Francisco, CA.
Ryan, I.P. and Taylor, R.N. (1997) Endometriosis and infertility: new concepts. Obstet. Gynecol. Surv., 52, 365371.[Medline]
Sharpe-Timms, K.L. (1997) Basic research in endometriosis. Obstet. Gynecol. Clin. North Am., 24, 269290.[ISI][Medline]
Sharpe, K.L. and Vernon, M.W. (1993) Polypeptides synthesized and released by rat endometriotic tissue differ from those of the uterine endometrium in culture. Biol. Reprod., 48, 13341340.[Abstract]
Sharpe, K.L., Zimmer, R.L., Griffin, W.T. et al. (1993) Polypeptides synthesized and released by human endometriosis tissue differ from those of the uterine endometrium in culture. Fertil. Steril., 60, 839851.[ISI][Medline]
Sharpe-Timms, K.L., Bruno, P.L., Penney, L.L. et al. (1994) Immunohistochemical localization of granulocyte-macrophage colony stimulating factor (GM-CSF) in matched endometriosis and endometrial tissues. Am. J. Obstet. Gynecol., 171, 450455.
Sharpe-Timms, K.L., Piva, M., Ricke, E.A. et al. (1998) Endometriotic lesions synthesize and secrete a haptoglobin-like protein. Biol. Reprod., 58, 988994.[Abstract]
Sharpe-Timms, K.L., Zimmer, R.L., Ricke, E.A. and Horowitz, G.M. (2000a) Aberrant adherence of peritoneal macrophages in endometriosis [Abstract O-57]. In Proceedings of the American Society of Reproductive Medicine, San Diego, CA.
Sharpe-Timms, K.L., Piva, M. and Ricke, E.A. (2000b) Extrahepatic vs. hepatic haptoglobin in women with endometriosis [Abstract P-825]. In Proceedings of the Society of Gynecological Investigations., Chicago, IL.
Sugawara, J., Yoshida, H., Fukaya, T. et al. (1997) Increased secretion of hepatocyte growth factor by eutopic endometrial stromal cells in women with endometriosis. Fertil. Steril., 68, 468472.[ISI][Medline]
Taylor, R.N., Ryan, I.P., Moore, E.S. et al. (1997) Angiogenesis and macrophage activation in endometriosis. Ann. N.Y. Acad. Sci., 828, 194207.[Abstract]
Tseng, J.F., Ryan, I.P., Milam, T.D. et al. (1996) Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis. J. Clin. Endocrinol. Metab., 81, 11181122.[Abstract]
Turner, G.A. (1995) Haptoglobin. A potential reporter molecule for glycosylation changes in disease. Adv. Exp. Med. Biol., 376, 231238.[Medline]
van Dijk, W., Turner, G.A. and Mackiewicz, A. (1994) Changes in glycosylation of acute-phase proteins in health and disease: occurrence, regulation and function. Glycosylation and Disease, 1, 514.
Vernon, M.W. and Wilson, E.A. (1985) Studies on the surgical induction of endometriosis in the rat. Fertil. Steril., 44, 684694.[ISI][Medline]
Wingfield, M., Macpherson, A., Healy, D.L. et al. (1995) Cell proliferation is increased in the endometrium of women with endometriosis. Fertil. Steril., 64, 340346.[ISI][Medline]
Submitted on April 18, 2000; accepted on June 21, 2000.