Human Reproduction vol.6 no 10 pp.1470-1473, 1991

Expression of intermediate filament in endoometrial glands changes with the onset of pregnancy and in endometriosis

E.R.Norwitz1, S.Fernandez-Shaw, D.H.Barlow and P.M.Starkey University of Oxford, Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK 'To whom correspondence should be addressed

Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermediate filament proteins, cytokeratin and vimentin, in human endometrium and first trimester decidua and in ectopic endometrium from women with endometriosis. Stromal elements, including vascular endothelial cells, were consistently vimentin-positive and cytokeratin-negative. Surface and glandular epithelial cells of human endometrium co-expressed vimentin and cytokeratin during all stages of the menstrual cycle, but failed to express vimentin after the onset of pregnancy. This suggests that intermediate filaments, and especially vimentin, may have a role to play in the proliferation and/or differentiation of the endometrial glands during decidualization. Ectopic endometrium showed a staining pattern similar to normal endometrium. Key words: decidua/endometrial glands/endometriosis/human/ vimentin Introduction Intermediate filaments constitute a distinct fibrous network within the cytoplasm of higher eukaryotic cells, and can be divided into five biochemically and immunologically distinct subclasses: cytokeratin, desmin, vimentin, glial and neurofilaments. In this study we have set out to investigate, using immunohistology of frozen tissue sections, the expression of two of the intermediate filament subclasses, namely cytokeratin and vimentin, in human endometrium (both normal and ectopic) and first trimester decidua.

Materials and methods Tissue samples First trimester decidual tissue (« = 6) of gestational age 7—12 weeks (calculated from the first day of the last menstrual period) was identified morphologically from materials obtained at therapeutic abortion. Blocks of endometrium/myometrium were obtained between days 5 and 22 of the menstrual cycle from 1470

the uterine cavity of pre-menopausal women undergoing hysterectomy for reasons other than malignancy. The stage was confirmed by histological analysis (Noyes et al., 1950); eight were proliferative phase and six secretory phase. Visible endometriotic implants (n = 6; one ovary, one serosal surface of the uterus, one peritoneum, two ovarian cyst wall, one uterosacral ligament) were biopsied at the time of diagnostic laparoscopy in patients (n = 4) with endometriosis, none of whom had received hormonal or other treatment in the 3-month period prior to surgery. At the time of biopsy, three of these women were in the secretory phase (days 16—19) and one in the proliferative phase (day 7). All samples for immunohistochemistry were snap-frozen in liquid nitrogen and stored at — 70°C. Tissues were collected in accordance with the requirements of the Central Oxfordshire Research Ethics Committee. Immunohistochemical studies Cryostat sections ( 5 - 7 jxm) were cut, acetone-fixed and stained using an indirect immunoperoxidase technique as previously described (Sutton et al., 1986). Briefly, tissue sections were pre-treated with 40 y\ non-immune rabbit serum (Sigma Chemical Co., Dorset, UK) to saturate non-specific binding sites, and stained with primary antibody: JMB-2 which labels cytokeratin no. 18 (Professor McGee, Oxford, UK; McGee et al., 1982), V9 (Dako Ltd, Bucks, UK; Osborn et al., 1984) or Vim 3B4 (Boehringer Mannheim Biochemical, Sussex, UK), both of which react with vimentin. Peroxidase-conjugated rabbit-anti-(mouse IgG) IgG (Dako) was added and the reactivity of each of the antisera was visualized using diaminobenzidine tetrahydrochloride (DAB; Sigma). Positive staining was confirmed by a clearly visible brown intracellular DAB precipitate. Negative controls used mouse-IgG (Coulter Electronics Ltd, Bedfordshire, UK) in place of the first antibody or peroxidase-conjugated second antibody only. Results Non-pregnant endometrium Both glandular and stromal cellular components of human endometrial tissues were found to be positive for vimentin (V9 and Vim 3B4 giving identical staining patterns). Staining of the glandular cells was most intense in the apical region. Surface and glandular epithelial cells stained positive for cytokeratin (JMB-2), but stromal elements including vascular endothelium were cytokeratin-negative (Figure la and b). There was no antibody binding in the negative control tissue sections (Figure lc). The staining pattern was consistent throughout the menstrual © Oxford University Press

Intermediate filament expression in endometriuin

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r Fig. 1. Immunohistology of human endometrium and first trimester decidua with antibodies to cytokeratin and vimentin (magnification X250). Frozen tissue sections were of secretory endometrium, day 10 (a—c) or first trimester decidua, week 6 (d-f)- Sections were labelled with antibody to vimentin (a,d), cytokeratin (b,e) or with mouse IgG as control (c,f). Bound antibody was detected with peroxidase-conjugated second antibody, the sections were counterstained with haematoxylin.

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cycle and no difference in staining could be discerned between the stratum basalis and the stratum functionalis in each patient. First trimester decidual tissue The surface and glandular epithelium of human first trimester decidua stained positive for cytokeratin but failed to bind antivimentin antibody. Stromal elements were cytokeratin-negative and vimentin-positive (Figure Id and e). This was consistent in all specimens examined. Negative control tissue sections had no detectable antibody binding (Figure If). No difference in staining pattern was noted between samples of differing gestational age, or between the decidua basalis as compared to the decidua functionalis. Endometriotic tissue Microscopic examination confirmed the presence of endometrial glandular and stromal elements in all biopsy specimens. In all sections examined, glandular epithelial cells were cytokeratinpositive and stromal cells cytokeratin-negative. The staining pattern seen with anti-vimentin antibody, however, was somewhat variable. Stromal elements were consistently vimentinpositive, while the glandular epithelial cells were positive in four but negative in one section. The remaining section showed evidence of both positive and negative reactivity in the glands (not shown). Discussion Our results confirm the co-expression of vimentin and cytokeratin intermediate filaments in the glandular and surface epithelial cells of normal human endometrium throughout the menstrual cycle (McNutt et al., 1985; Viale et al., 1988; Ohwada et al., 1990). After the onset of pregnancy, however, the glands and surface epithelium of first trimester decidua consistently failed to express vimentin. Intermediate filaments are usually expressed exclusively in any given differentiated cell type (Puts et al., 1986), although the coexistence of two different intermediate filament subunits in a single cell type has been previously documented and usually includes vimentin as one of them (McNutt et al., 1985; Viale etal., 1988; Osteen et al., 1989). Vimentin is a 57 kDa intermediate filament protein present in the cytoplasm of cells of mesenchymal origin, but it is also expressed in fetal cells (Van Muijen et al., 1987) and in cells in culture (Lazarides, 1982), irrespective of their tissue of origin. Cytokeratins, on the other hand, are found in cells of epithelial origin. The importance of intermediate filament co-expression remains unclear. In preparation for pregnancy, human endometrium undergoes both physiological (Maathuis and Kelly, 1978; Eddie et al., 1986; Bryant-Greenwood etal., 1987) and morphological changes (Bulmer and Sunderland, 1984; Starkey et al., 1988; Aplin et al., 1988), which are loosely referred to as 'decidualization'. While alterations in membrane antigen expression by endometrial glands during this period are well described (Johnson and Bulmer, 1984), changes in cytoplasmic antigen expression are less well recognized. Ohwada et al. (1990) reported stronger vimentin reactivity during the proliferative phase of the menstrual cycle but our results failed to demonstrate any variation. Since the 1472

function(s) of intermediate filaments in the cell are poorly understood, the loss of vimentin expression in human endometrial glands with the onset of pregnancy is difficult to explain. It has been suggested that intermediate filaments serve primarily a structural function in the cytoplasm (Lehto and Virtanen, 1978); the loss of one of these cytoskeletal proteins in the endometrial glands might make them more susceptible to implantation. Alternatively, the loss of vimentin expression may reflect a change in glandular function since its depletion has been associated with an alteration in the levels of endometrial secretory component (Nakopoulou et al., 1990). Functional changes which have been associated with the onset of pregnancy include an increase in the secretion of relaxin (Eddie et al., 1986) and prolactin (Bryant-Greenwood et al., 1987) as well as a decrease in prostaglandin production (Maathuis and Kelly, 1978). In general, the staining pattern seen in ectopic endometrium was similar to that seen in normal uterine endometrium, which is in agreement with recent publications (Matthews et al., 1991). In our study, however, vimentin reactivity with epithelial glands of ectopic endometrium showed some variability. The significance of this observation remains unclear but may be related to differences in the levels of secreted proteins compared to glands of uterine endometrium (Kauppila et al., 1984). This hypothesis requires further investigation.

Acknowledgements Professor McGee is thanked for his gift of JMB-2 antibody. Dr E.R.Norwitz is a Rhodes Scholar. This work was supported by the Rhodes Trust. Dr S.Fernandez-Shaw is supported by a Florey E.P.Abrahams European Studentship (Queen's College, Oxford).

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J. Endocrinol., 114, 491-4%. Bulmer,J.N. and Sunderland, C. A. (1984) Immunohistological characterisation of lymphoid cell populations in the early human placenta] bed. Immunology, 52, 349-357. Eddie.L.W., Bell.R.J., Lester.A., Geier,M., Bennett,G., Johnston.P.D. and Niall.H.D. (1986) Radioimmunoassay of relaxin in pregnancy with an analogue of human relaxin. Lancet, i, 1344 — 1346. Johnson,P.M. and Bulmer,J.N. (1984) Uterine gland epithelium in human pregnancy often lacks detectable maternal MHC antigens but does express fetal trophoblast antigens. J. Immunol., 132, 1608-1610. Kauppila.A., Vierikko.P., Isotalo.H., R6nnberg,L. and Vihko,R. (1984) Cytosol estrogen and progestin receptor concentrations and 17 /3-hydroxysteroid dehydrogenase activities in the endometrium and endometriotic tissue. Effects of hormonal treatment. Acta. Obsiei. Gynaecol. Scand., (Suppl.) 123, 45-49. Lazarides,E. (1982) Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu. Rev. Biochem.. 51, 219-250.

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Lehto,V.P. and Virtanen.I. (1978) Intermediate filaments anchor the nuclei in nuclear monolayers of cultured human fibroblasts. Nature, 272, 175-177. Maathuis,J.B. and Kelly,R.W. (1978) Concentrations of prostaglandins F 2a and Ej in the endometrium throughout the human menstrual cycle after the administration of clomiphene or an oestrogenprogesterone pill and in early pregnancy. J. Endocrinol., 77, 361-371. Matthews.C.J., Redfern.C.P.F., Hirst.B.H. and Thomas,E.J. (1991) Culture of endometrium and endometriosis: characterisation of cells by cytoskeletal components and epithelial mucins. J. Reprod. Fertil., Abstr. Ser. No. 7, 41, (Abstract No. 72). McGeeJ.O'D., MortonJ.A., Barbatis.C, Bradley,J.F., Fleming,K.A. Goate.A.M. and Brons,J. (1982) Monoclonal antibodies/intermediate filaments and HLA (class I) antigens in human liver disease. In McMichael,A.J. and FabreJ.W. (eds), Monoclonal Antibodies in Clinical Medicine. Academic Press, London, pp. 431-455. McNutt,M.A., Bolen.J.W., Gown,A.M., Hammar.S.P. and Vogel,A.M. (1985) Co-expression of intermediate filaments in human epithelial neoplasms. Ultrastruct. Pathol., 9, 31—43. Nakopoulou,L., Minaretzia.D., Tsionou,C. and Mastrominas.M. (1990) Value of immunohistochemical demonstration of several epithelial markers in hyperplasia and neoplastic endometrium. Gynaecol. Oncol., 37, 346-353. Noyes.R.W., Hertig.A.T. and Rock,J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3 - 2 5 . Ohwada,M., Suzuki,M. andTamada.T. (1990) [Immunohistochemical investigation of vimentin of uterine endometrial and endocervical adenocarcinoma]. Nippon-Sanka-Fujinka-Gakkai-Zasshi, 25, 1454-1460. Osborn,M., Debus,E. and Weber.K. (1984) Monoclonal antibodies specific for vimentin. Eur. J. Cell. Biol., 334, 137-143. Osteen,K.G., Hill.G.A., Hargrove,J.T. and Gorstein.F. (1989) Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens. Fertil. Steril., 52, 965-972. Puts,J.J., Vooijs.G.P., Huysmans,A., van Aspert,A. and Ramaekers, F.C. (1986) Cytoskeletal proteins as tissue-specific markers in cytopathology. Exp. Cell. Biol., 54, 73-79. Starkey.P.M., Sargent,I.L. and Redman.C.W.G. (1988) Cell populations in human early pregnancy decidua: characterisation and isolation of large granular lymphocytes by flow cytometry. Immunology, 65, 129-134. Sutton.L., Gadd,M., Mason,D.Y. and Redman.C.W.G. (1986) Cells bearing class II MHC antigens in the human placenta and amniochoron. Immunology, 58, 23—29. Van Muijen,G.N., Ruiter,D.J. and Wamaar.S.O. (1987) Coexpression of intermediate filament polypeptides in human fetal and adult tissues. Lab. Invest., 57, 359-369. Viale,G., Gambacorta.M., Dell'Orto,P. and Coggi.G. (1988) Coexpression of cytokeratins and vimentin in common epithelial tumours of the ovary: an immunohistochemical study of eighty-three cases. Virchows Arch. [A.] Pathol. Anat., 413, 91-101. Received on May 13, 1991; accepted on August 14, 1991

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Expression of intermediate filament in endometrial glands changes with the onset of pregnancy and in endometriosis.

Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermed...
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