Human Reproduction vol.7 no.9 pp. 1189-1194, 1992
Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells
Department of Gynaecology and Obstetrics, Faculty of Medicine and 2Chest Disease Research Institute, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan 'To whom correspondence should be addressed
To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistotogica] studiesrevealedthat DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proUferative phase, began to increase gradually in the early secretory phase, and was strong in midto late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histocbemkally in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proUferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter. Key words: dipeptidyl peptidase FV/endometria] glandular cells/endometrial surface epithelium/enzyme histochemistry/ immunohistology
autocrine systems involving these substances have been suggested (Yagel et al., 1989; Kauma et al., 1990, 1991; Giudice et al., 1991; Hofmann etal., 1991; Seppala etal., 1991; Strowitzki etal., 1991; Tabibzadeh, 1991). Recently, we have reported (Imai et aL, 1992) that endometrial stromal cells and decidual cells of the first trimester of pregnancy, express both the cluster of differentiation (CD) 13 and CD 10 antigens, which are identical to aminopeptidase N (Look et al., 1989) and neutral endopeptidase (Letarte et al., 1988), respectively. We also showed that these two peptidases were useful cell surface markers of endometrial stromal cells and decidual cells and that an endometrial stromal cell-enriched preparation had peptidase activity (Imai et al., 1992). As these two peptidases are membrane-bound ecto-enzymes, which expose their catalytic sites on the cell surface, various kinds of biologically active peptides can be degraded which are present extracellularly (Erdos and Skidgel, 1989; Ward et al., 1990). We therefore proposed that membrane-bound ecto-enzymes could regulate the local concentration of biologically active peptides at the materno—fetal interface to achieve successful implantation and maintenance of pregnancy. The involvement of CD 13 antigen/aminopeptidase N in the follicular development of the human ovary was also suggested (Fujiwara et al., 1992). These findings prompted us to investigate whether another cell surface peptidase, namely dipeptidyl peptidase (DPP) IV/CD26 antigen, was expressed in the human endometrium. Here we report that human endometrial glandular cells and endometrial surface epithelium express DPP IV/CD26 antigen, as detected by immunohistology and enzyme histochemistry. Materials and methods
Introduction
Tissues Human endometria were obtained from patients who had undergone hysterectomy for the treatment of uterine myoma, uterine prolapse or adenomyosis. First trimester human deciduae were obtained from patients who had undergone legal abortions. Informed consent was obtained from every patient. Gestational age was calculated from the date of the last menstrual period and, if necessary, was adjusted by taking into account the ultrasonic measurement of the gestational sac and fetal crown—rump length.
Human endometrium is composed mainly of two types of cells, endometrial glandular cells and endometrial stromal cells, of which the growth and differentiation are under ovarian sexsteroidal control. Accumulating evidence shows that different kinds of biologically active peptides, cytokines and peptide growth factors, are present in the human endometrium, and paracrine/
Antibodies A mouse monoclonal antibody, Tal(CD26, anti-DPP IV) (Fox et al., 1984; Ulmer et al., 1990) was obtained from Coulter Immunology (Hialeah, FL, USA), and was used at a dilution of 1:100. The specificity of Tal to DPP IV has been previously
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Kimitoshi Imai1, Michiyuki Maeda2, Hirosbi Fujiwara, Masatoshi Kariya, Kenji Takakura, Hideharu Kanzaki and Takahide Mori
K.lmai et al.
reported (Mattern et al., 1989; Stein et al., 1989). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dakopatts A/S, Glostrup, Denmark) was used as a developing antibody at a dilution of 1:40. Indirect immunofluorescence staining
Fig. 1. Indirect immunofluorescence staining of human endometrium with anti-dipeptidyl peptidase (DPP) IV monoclonal antibody, (a) Midproliferative phase; (b,e) mid-secretory phase; (c,f) late secretory phase; (d) the first trimester of pregnancy (6 weeks of gestation). DPP IV expression on endometrial glandular cells (GL) was weak in proliferative phase (a) whereas the expression was strong in mid- (b) and late (c) secretory phase and during the first trimester of pregnancy (d). Endometrial surface epithelium (SE) also showed strong expression of DPP IV in the mid-secretory phase (e). Endometrial stromal cells (ESC) and decidual cells (DC) did not express DPP IV ( a - f ) . Apical membrane of GL and SE appeared to express DPP IV stronger than the basolateral membranes (b-f). Heterogeneity of DPP IV expression in GL was shown in (f). a-f: Magnification X140.
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Fresh tissue samples were cut into small pieces, embedded in OCT compound (Miles Inc., Elkhart, IN, USA), frozen with Liquid nitrogen, and kept at -80°C until sectioned. Cryostat (Reichert-Jung, Heidelberg, FRG) sections, ~ 6 /tm thick, were air-dried and fixed with acetone at -20°C for 5 min. Non-
specific immunoglobulin binding was blocked by preincubation with 5% normal rabbit serum in phosphate-buffered saline (PBS, pH 7.4) for 15 min. The sections were then incubated for 1 h with the monoclonal antibody in a moist chamber at room temperature. After three washes with PBS, they were incubated for 30 min with FITC-conjugated rabbit anti-mouse immunoglobulin. After another three washes with PBS, the sections were examined with a fluorescence microscope (Nikon, Tokyo, Japan). For negative controls, the first antibody was replaced by normal mouse serum at an appropriate dilution. One cryostat section of each sample was stained with
Dipeptidyl peptidase IV In the human endometrium
haematoxylin and eosin, and was examined morphologically with a light microscope.
Results Indirect immunoflwrescence staining Endometria were obtained from 23 women, 35 to 48 years old, with regular menstrual cycles and deciduae were obtained from 11 women, 18 to 40 years old, at 6 —13 weeks of gestation. Ten endometria were in proliferative phase, four were in early secretory phase, four were in mid-secretory phase, and five were in late secretory phase. As shown in Figure 1 and Table I, DPP IV was expressed on endometrial glandular cells and surface epithelium. Judging from the fluorescence intensity, the expression of DPP IV on both these areas appeared weak during the proliferative phase (Figure la). The staining of DPP IV was stronger in the early secretory phase than in the proliferative phase (data not shown). In the mid- to late secretory phase, and in the first trimester of pregnancy, DPP IV expression was strong (Figure l b - e ) . It appeared that DPP IV expression on the apical membrane of glandular cells and surface epithelium was stronger than that on the basolateral membrane (Figure l b - f ) . Glandular cells in the basal layer of the endometrium also appeared to show the cyclic change of the DPP IV expression, although the expression was weaker than that in the functional layer (data not shown). In three
Histochemistry The endometria of 28 women, 37 and 49 years old, and nine first trimester deciduae, at 6 — 13 weeks gestation, were examined. Of the 28 endometria, 10 were in the proliferative phase, five were in early secretory phase, six were in midsecretory phase, and seven were in late secretory phase (Table H). DPP IV enzyme activity was rarely detected in endometrial glandular cells and surface epithelium in the proliferative and early secretory phases (Figure 2a), but in the mid-secretory phase, DPP IV activity became detectable (Figure 2b). In the late secretory phase, glandular cells and surface epithelium had strong DPP IV activity (Figure 2c). During the first trimester of pregnancy, DPP IV activity in both areas was slightly weaker than that in the late secretory phase (Figure 2d). There appeared to be no difference of DPP IV activity between glandular cells and surface epithelium (Figure 2b). The apical membranes of glandular cells and of surface epithelium appeared to show stronger DPP IV activity than the basolateral membranes (Figure 2c). Glandular cells in the basal layer had DPP IV enzyme activity in the mid-secretory phase and thereafter, but the activity appeared weaker than that in glandular cells of the functional layer (data not shown). In some samples in mid- and late secretory phases, DPP IV enzyme activity in the glandular cells was heterogeneous (Figure 2b). By contrast, endometrial stromal and decidual cells and other cells in the endometrial stroma did not have DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy, in any of the specimens which we have examined (Figure 2a -d). Although the endothelium of some vessels in the uterine muscle layer showed weak DPP IV activity, the endothelium of the vessels in the endometrium did not show any detectable DPP IV activity (data not shown). Uterine muscle did not show DPP IV enzyme activity (data not shown).
Table I. Immunohistological detection of dipeptidyl peptidase (DPP) IV Proliferative phase [n = 10) GL SE ESC/DC
Secretory phase Early (n = 4)
Mid (n = 4)
Late (n = 5)
First trimester of pregnancy (n = 11)
+- ++
+
+++
+H
+
h++
h+ +
GL = endometrial glandular cells; SE = endometrial surface epithelium, ESC = endometrial stromal cells; DC = decidual cells. Immunofluorescence intensity was arbitrarily graded as: — , negative; + weakly positive; + + , moderately positive; and + + + , strongly positive.
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Histochemical staining Unfixed cryostat sections, ~ 6 /on thick, were air dried, and kept at —80cC until assay, which was performed within 2 weeks. The enzyme-histochemical reaction for DPP IV was carried out according to the method of Lojda (1979), with minor modifications. Briefly, 4 mg of glycyl-prolyl-4-methoxy-/?naphthylamide (Bachem, Bubendorf, Switzerland) was dissolved in 0.5 ml N,N-dimethylformamide (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and mixed with 10 mg Fast Blue B salt (Sigma Chemical Company, St Louis, MO, USA) dissolved in 10 ml 0.1 M phosphate buffer, pH 7.3. The mixture was filtered through a 0.22 y.m membrane filter and immediately overlaid on slides. Incubation was carried out at room temperature for 6—7 min. After incubation, the sections were washed in distilled water, air dried, and observed with a light microscope. Glycyl-prolyl-4-methoxy-/3-naphthylamide has been reported to be a specific substrate for DPP IV in histochemistry (Lodja, 1979). As negative controls, cryostat sections were incubated with substrate-free media (Hartel etai, 1988).
out of nine mid- and late secretory phase endometrial samples, the grade of DPP IV expression on the glandular cells differed greatly from gland to gland in the functional layer, despite there being no obvious microscopic morphological differences between the glands (Figure If)By contrast, endometrial stromal and decidual cells, and other cells in the endometrial stroma, did not express DPP IV at any time throughout the menstrual cycle or during the first trimester of pregnancy (Figure l a - f ) . Although the endothelium of some vessels in the uterine muscle layer expressed DPP IV, the endothelium of vessels in the endometrium did not appear to express DPP IV (data not shown). Uterine muscle did not express DPP IV (data not shown).
K.Imai etal.
Table U. Enzyme-histochemical activity of dipeptidyl peptidase (DPP) IV ProUferative phase (n -10)
Secretory phase Early (n = 5)
Mid (n = 6)
+ ~+-f
GL SE ESC/DC
Late (n = 7)
:r
First trimester of pregnancy (n — 9)
I- + +
SE
GL
Fig. 2. Enzyme histochemistry showing dipeptidyl peptidase (DPP) IV activity in the human endometrium. (a) Proliferative phase; (b) midsecretory phase, (c) late secretory phase; (d) during the first trimester of pregnancy (8 weeks of gestation). DPP IV activity on endometrial glandular cells (GL) was rarely detected in the proliferative phase endometrium (a). DPP IV activity in GL and endometrial surface epithelium (SE) became detectable in the mid-secretory phase (b), and was strong in the late secretory phase (c) and during the first trimester of pregnancy (d). Endometrial stromal cells (ESQ and decidual cells (DQ did not express DPP IV activity during the menstrual cycle or the first trimester of pregnancy (a—d). DPP IV activity appeared stronger on the apical membrane than on the basolateral membranes of GL and SE (c). a,b and d; Magnification X70; c: magnification x 140.
Discussion The results presented in this paper can be summarized as follows. Firstly, on indirect immunofluorescence staining, both endometrial glandular cells and endometrial surface epithelium, but not endometrial stromal cells or decidual cells, express DPP FV/CD26 antigen throughout the menstrual cycle and during the first trimester of pregnancy. The immunoreactivity is weak in the proliferative phase, gets stronger in the early secretory phase, and remains strong in the mid- to late secretory phase, and in the first trimester of pregnancy. Secondly, histochemically, DPP IV enzyme activity is detected in endometrial glandular cells and 1192
endometrial surface epithelium in the mid-secretory phase and thereafter. Endometrial stromal cells and decidual cells show no detectable DPP IV enzyme activity. These findings are in parallel with those obtained by indirect immunofluorescence staining. Thirdly, the immunoreactivity and enzyme activity of DPP IV is stronger on the apical membrane of the endometrial glandular cells and endometrial surface epithelium than on the basolateral membranes. These findings indicate firstly that DPP IV is an excellent cell marker of endometrial glandular cells and endometrial surface epithelium; secondly that DPP IV can be considered to be a
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GL = endometrial glandular cells; SE = endometrial surface epithelium; ESC = endometrial stromal cells; DC = decidual cells. •Weakly positive GL were also sporadically noted in two out of 10 specimens. The staining was arbitrarily graded as: —, negative; + , weakly positive; + + , moderately positive; + + + , strongly positive.
Dipeptidyl peptidase IV in the human endometriom
antigen was shown to be identical to DPP IV (Mattern et al., 1989; Stein etal., 1989; Ulmer etal., 1990). This antigen is expressed strongly on activated T lymphocytes and on some resting T lymphocytes (Fox et al., 1984). Inhibition of DPP IV enzyme activity is shown to suppress T lymphocyte function (Schon et al, 1989; Flentke et al., 1991). Therefore DPP IV is considered to play an important role(s) in the immune system. Similarly, DPP IV on the endometrial glandular cells and on the endometrial surface epithelium might play some immunological role(s). Furthermore, DPP IV has been shown to be involved in cellular adhesion in extracellular matrix proteins (Hanski et al., 1985, 1988; Piazza et al., 1989; Dang et al., 1990). Since DPP IV is expressed on the apical membrane of endometrial surface epithelium during the peri-implantation period, this antigen might affect the attachment of fertilized eggs on to the endometrial surface. In this regard, Denker (1977) previously suggested the importance of proteinases in implantation. In conclusion, DPP IV is established as a differentiation marker for human endometrial glandular cells and endometrial surface epithelium. The role(s) of DPP IV in human reproduction remains to be clarified.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (no. 03454396) from the Ministry of Education, Science and Culture of Japan and by the Shimizu Foundation Research Grant for 1990.
References Bulmer,J.N. and Johnson,P.M. (1985) Immunohistological characterization of the decidual leukocytic infiltrate related to endometrial gland epithelium in early human pregnancy. Immunology, 55, 35-44. Classen-Linke,I., Denker,H.-W. and Winterhager,E. (1987) Apical plasma membrane-bound enzymes of rabbit uterine epithelium. Pattern changes during the peri-implantation phase. Histochemistry, 87, 517-529. Dang.N.H., Torimoto.Y., Schlossman.S.F. and Morimoto.C. (1990) Human CD4 helper T cell activation: functional involvement of two distinct collagen receptors 1F7 and VLA integrin family. J. Exp. Med, Yll, 649-652. Denker,H.-W. (1977) Implantation. The role of proteinases, and blockage of implantation by proteinase inhibitors. Adv. Anal. Embryol. Cell Bioi, 53, 1-123. Erdos.E.G. and Skidgel.R.A. (1989) Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J., 3, 145-151. Flentke.G.R., Munoz.E., Hubner,B.T., Plaut,A.G., Kettner.C.A. and Bachovchin.W.W. (1991) Inibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function. Proc. Nail. Acad. Set. USA, 88, 1556-1559. Fox,D.A., Hussey.R.E., Fitzgerald.K.A., Acuto.O., Poole.C, Palley,L., DaleyJ.F., Schlossman.S.F. and Reinherz.E.L. (1984) Tal, a novel 105 kD human T cell activation antigen defined by a monoclonal antibody. J. Immunol., 133, 1250—1256. Frohmann,L.A., Downs.T.R., Heimer.E.P. and Felix.A.M. (1989) Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma. J. CUn. Invest., 83, 1533-1540.
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differentiation antigen for endometrial glandular cells and endometrial surface epithelium, since immunoreactivity and enzyme activity due to DPP IV are dependent on the stages of endometrial differentiation; and thirdly that biologically active DPP IV is present on endometrial glandular cells and on endometrial surface epithelium during the peri-implantation period and thereafter. It is well established that lymphocytes, granulocytes and macrophages are distributed in the human endometrium and decidua (Buhner and Johnson, 1985; Kamat and Isaacson, 1987), and DPP IV is shown to be present in some T lymphocytes (Lojda, 1977; Fox et al., 1984; Schon et al, 1984). However, our results showed that blood cells in the endometrium as well as endometrial stromal and decidual cells had no detectable DPP IV immunoreactivity or enzyme activity. Lojda (1979) reported that, histochemically, DPP IV activity in the endothelium of blood vessels was heterogeneous. In our study most of the endothelium in the uterine muscle layer was DPP IV-positive, but the endothelium in the endometrium did not show DPP IV, either histochemically or immunohistologically. This finding is consistent with Lojda's observation. DPP IV has been extensively studied both biochemically and histochemically, in various animals and in humans (Hopsu-Havu and Glenner, 1966; Gossrau, 1979; Heyman and Mentlein, 1984; Hartel et al., 1988; Heike et al., 1988). The presence of DPP IV in endometrial glandular cells and in the endometrial surface epithelium has been reported in the rat (Gossrau, 1979) and rabbit (Classen-Linke et al, 1987). Classen-Linke et al. (1987) reported changes in DPP IV activity on the endometrial epithelium during the peri-implantation period in a study using pseudopregnant and pregnant rabbits, and suggested that the changes might be due to steroid hormones or other mediators that may be produced by blastocysts. DPP IV is a membrane-bound ecto-enzyme, which exposes its catalytic site on the cell surface and removes mainly Xaa-Pro dipeptides from the N-termini of polypeptides (Yoshimoto et al., 1978; Heyman and Mentlein, 1984; Nausch et al., 1990). Its possible substrates include human gastrin-releasing peptide, human pancreatic polypeptide (Nausch et al., 1990), substance P (Puschel etal., 1982), human growth hormone-releasing hormone (Frohmann et al., 1989), and others. DPP IV can also degrade the alpha chain of human chorionic gonadotrophin (Nausch et al., 1990), which is secreted by trophoblasts. This suggests that DPP IV could affect the local metabolism of biologically active peptides present extracellularly in the female genital tract. Kenny et al. (1989) suggested the possibility that cell surface peptidase may have a key role in the control of growth and differentiation of many cellular systems in vivo by modulating the activity of peptide factors and regulating their access to adjacent cells. We have shown that the expression of aminopeptidase N, neutral endopeptidase, and DPP IV in the human endometrium is menstrual cycle dependent. Therefore, our present findings provide supporting evidence for Kenny et al.'s proposal (1989). DPP IV is also known to be a T lymphocyte surface antigen. Fox et al. (1984) reported Tal as a novel T lymphocyte-activation antigen, and it has been classified as CD26 antigen. Later CD26
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Schmidt.R.E., Stein.H. and von dem Borne.A.E.G.K. (eds), Leukocyte Typing IV. White Cell Differentiation Antigens. Oxford University Press, Oxford, New York, Tokyo. Nausch.I., Mentlein.R. and Heymann.E. (1990) The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta. Biol. Chem. Hoppe-Seyler, 371, 1113-1118. Piazza.G.A., Callanan.H.M., Mowery.J. and Hixson.D.C. (1989) Evidence for a role of dipeptidyl peptidase IV in fibronectin-mediated interactions of hepatocytes with extracellular matrix. Biochem. J., 262, 327-334. Puschel.G., Mentlein.R. and Heymann.E. (1982) Isolation and characterization of dipeptidyl peptidase IV from human placenta. Eur. J. Biochem., 126, 359-365. Schon.E., Demuth,H.-U., Barth.A. and Ansorge.S. (1984) Dipeptidyl peptidase IV of human lymphocytes. Evidence for specific hydrolysis of glycylproline/>-nitroanilide in T-lymphocytes. Biochem. J., 223, 255-258. Schon.E., Demuth,H.-U., Eichmann.E., Horst,H.-J., K6rner,I.-J., KoppJ., Mattem.T., Neubert.K., Noll.F., Ulmer.A.J., Barth.A. and Ansorge.S. (1989) Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interieukin 2 and gamma interferon due to specific inhibition of dipeptidyl peptidase IV. Scand. J. Immunol., 29, 127-132. Seppala.M., Yajima.M., Koistinen,R., Angervo.M., Riittinen.L., Suikkari.A.M., Selenius.P. and Julkunen.M. (1991) Endometrial proteins as local regulators of human endometrial function and their appearance in serum: clinical applications. Ann. N. Y. Acad. Sci., 626, 312-320. Stein.H., Schwarting.R. and Niedobhek.G. (1989) Cluster report: CD26. In Knapp.W., Dorken.B., Gilks.W.R., Rieber.E.P., Schmidt.R.E., Stein.H. and von dem Borne.A.E.G.Kr. Leukocyte Typing IV. White Cell Differentiation Antigens. Oxford University Press, Oxford, New York, Tokyo, pp. 412-415. Strowitzki,T., Wiedemann.R. and Hepp.H. (1991) Influence of growth factors EGF, IGF-1, and human growth hormone on human endometrial stromal cells in vitro. Ann. N.Y. Acad. Sci., 626, 308-311. Tabibzadeh.S. (1991) Ubiquitous expression of TNF-a/cachectin immunoreactivity in human endometrium. Am. J. Reprod. Immunol., 26, 1-4. Ulmer.A.J., Mattern.T., Feller.A.C, Heymann.E. and Flad.H.D. (1990) CD26 antigen is a surface dipeptidyl peptidase IV (DPP IV) as characterized by monoclonal antibodies clone Til-19-4-7 and 4EL1C7. Scand. J. Immunol., 31, 429-435. Yagel.S., Lala.P.K., Powell.W.A. and Casper.R.F. (1989) Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast. / Clin. Endocrinol Metab., 68, 992-995. Yoshimoto.T., Fischl.M., Oriowski.R.C. and Walter.R. (1978) Postproline cleaving enzyme and post-proline dipeptidyl aminopeptidase. J. Biol. Chem., 253, 3708-3716. Ward.P.E., Benter.I.F., Dick.L. and Wilk.S. (1990) Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M. Biochem. Pharmacol, 40, 1725-1732. Received on April 22, 1992; accepted on July 2, 1992
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Fujiwara,H., Maeda,M., Imai.K., Fukuoka.M., Yasuda.K., Horie.K., Takakura.K., Taii.S. and Mori.T. (1992) Differential expression of aminopeptidase N on human ovarian granulosa cells and thecal cells. J. Clin. Endocrinol. Metab., 74, 9 1 - 9 5 . Giudice.L.C, Lamson.G., Rosenfeld.R.G. and IrwonJ. (1991) Insulinlike growth factor-II (IGF-II) and IGF binding proteins in human endometrium. Ann. N.Y., Acad. Sci., 626, 295-307. Gossrau.R. (1979) Peptidasen II. Zur Lokalization der Dipeptidylpeptidase IV (DPP IV). Histochemische und biochemische Untersuchung. Histochemistry, 60, 231-248. Hanski.C, Huhle.T. and Reutter.W. (1985) Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen. Biol. Chem. Hoppe-Seyler, 366, 1169-1176. Hanski.C., Huhle.T., Gossrau.R. and Reutter.W. (1988) Direct evidence for the binding of rat liver DPP IV to collagen in vitro. Exp. Cell. Res., 178, 6 4 - 7 2 . Hartel.S., Gossrau.R., Hanski.C. and Reutter.W. (1988) Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry. Histochemistry, 89, 151 — 161. Heike.M., Mobius.U., Knuth.A., Meuer.S. and Meyer Zum Buschenfelde,K.-H. (1988) Tissue distribution of the T cell activation antigen Tal. Serological, immunohistochemical and biochemical investigations. Clin. Exp. Immunol., 74, 431-434. Heyman.E. and Mentlein.R. (1984) Becinflu/3t Dipeptidylpeptidase IV Blutdruck und Gerinnung? Klin. Wochenschr., 62, 2 - 1 0 . Hofmann.G.E., Scott.R.T., Bergh,P.A. and Deligdisch,L. (1991) Immunohistochemical localization of epidermal growth factor in human endometrium, decidua, and placenta. J. Clin. Endocrinol. Metab., 73, 882-887. Hopsu-Havu,V.K. and Glenner.G.G. (1966) A new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-/3-naphthylamide. Histochemie, 7, 197-201. Imai.K., Maeda.M., Fujiwara.H., Okamoto.N., Kariya.M., Emi.N., Takakura.K., Kanzaki.H. and Mori.T. (1992) Human endometrial stromal cells and decidual cells express cluster of differentiation (CD)13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase. Biol. Reprod., 46, 328-334. Kamat.B.R. and Isaacson.P.G. (1987) The immunocytochemical distribution of leukocytic subpopulations in human endometrium. Am. J. Pathol., 127, 6 6 - 7 3 . Kauma.S., Matt.D., Strom.S., Eierman.D. and Tumer.T. (1990) Interieukin-1/}, human leukocyte antigen HLA-DRa, and transforming growth factor-/3 expression in endometrium, placenta, and placenta! membranes. Am. J. Obstet. Gynecol, 163, 1430-1437. Kauma.S.W., Aukerman,S.L., Eierman.D. and Turner.T. (1991) Colony-stimulating factor-1 and c-fins expression in human endometrial tissues and placenta during the menstrual cycle and early pregnancy. J. Clin. Endocrinol. Metab., 73, 746-751. Kenny.A.J., O'Hare.M.J. and Gusterson.B.A. (1989) Cell-surface peptidases as modulators of growth and differentiation. Lancet, 2, 785-787. Letarte.M., Vera.S., Tran.R., AddisJ.B.L., Onizuka.R.J. and Quackenbush.E.J. (1988) Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J. Exp. Med., 168, 1247-1253. Lojda.Z. (1977) Studies on glycyl-proline naphthylamidase. I. Lymphocytes. Histochemistry, 54, 299-309. Lojda.Z. (1979) Studies on dipeptkryl(amino)peptklase IV (grycyl-proline naphthylamidase). n. Blood vessels. Histochemistry, 59, 153-166. Look.A.T., Ashmun.R.A., Shapiro.L.H. and Peiper.S.C. (1989) Human myeloid plasma membrane glycoprotein CD13(gpl5O) is identical to aminopeptidase N. J. Clin. Invest., 83, 1299-1307. Mattem.T., Fled,H.-D., Feller.A.C, Heymann.E. and Ulmer.A.J. (1989) Anti-DPP-IV and anti-Tal but not IOT 15 should be clustered in CDw26. In Knapp.W., Dorken.B., Gilks.W.R., Rieber.E.P.,
Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells
Department of Gynaecology and Obstetrics, Faculty of Medicine and 2Chest Disease Research Institute, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan 'To whom correspondence should be addressed
To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistotogica] studiesrevealedthat DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proUferative phase, began to increase gradually in the early secretory phase, and was strong in midto late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histocbemkally in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proUferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter. Key words: dipeptidyl peptidase FV/endometria] glandular cells/endometrial surface epithelium/enzyme histochemistry/ immunohistology
autocrine systems involving these substances have been suggested (Yagel et al., 1989; Kauma et al., 1990, 1991; Giudice et al., 1991; Hofmann etal., 1991; Seppala etal., 1991; Strowitzki etal., 1991; Tabibzadeh, 1991). Recently, we have reported (Imai et aL, 1992) that endometrial stromal cells and decidual cells of the first trimester of pregnancy, express both the cluster of differentiation (CD) 13 and CD 10 antigens, which are identical to aminopeptidase N (Look et al., 1989) and neutral endopeptidase (Letarte et al., 1988), respectively. We also showed that these two peptidases were useful cell surface markers of endometrial stromal cells and decidual cells and that an endometrial stromal cell-enriched preparation had peptidase activity (Imai et al., 1992). As these two peptidases are membrane-bound ecto-enzymes, which expose their catalytic sites on the cell surface, various kinds of biologically active peptides can be degraded which are present extracellularly (Erdos and Skidgel, 1989; Ward et al., 1990). We therefore proposed that membrane-bound ecto-enzymes could regulate the local concentration of biologically active peptides at the materno—fetal interface to achieve successful implantation and maintenance of pregnancy. The involvement of CD 13 antigen/aminopeptidase N in the follicular development of the human ovary was also suggested (Fujiwara et al., 1992). These findings prompted us to investigate whether another cell surface peptidase, namely dipeptidyl peptidase (DPP) IV/CD26 antigen, was expressed in the human endometrium. Here we report that human endometrial glandular cells and endometrial surface epithelium express DPP IV/CD26 antigen, as detected by immunohistology and enzyme histochemistry. Materials and methods
Introduction
Tissues Human endometria were obtained from patients who had undergone hysterectomy for the treatment of uterine myoma, uterine prolapse or adenomyosis. First trimester human deciduae were obtained from patients who had undergone legal abortions. Informed consent was obtained from every patient. Gestational age was calculated from the date of the last menstrual period and, if necessary, was adjusted by taking into account the ultrasonic measurement of the gestational sac and fetal crown—rump length.
Human endometrium is composed mainly of two types of cells, endometrial glandular cells and endometrial stromal cells, of which the growth and differentiation are under ovarian sexsteroidal control. Accumulating evidence shows that different kinds of biologically active peptides, cytokines and peptide growth factors, are present in the human endometrium, and paracrine/
Antibodies A mouse monoclonal antibody, Tal(CD26, anti-DPP IV) (Fox et al., 1984; Ulmer et al., 1990) was obtained from Coulter Immunology (Hialeah, FL, USA), and was used at a dilution of 1:100. The specificity of Tal to DPP IV has been previously
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Kimitoshi Imai1, Michiyuki Maeda2, Hirosbi Fujiwara, Masatoshi Kariya, Kenji Takakura, Hideharu Kanzaki and Takahide Mori
K.lmai et al.
reported (Mattern et al., 1989; Stein et al., 1989). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dakopatts A/S, Glostrup, Denmark) was used as a developing antibody at a dilution of 1:40. Indirect immunofluorescence staining
Fig. 1. Indirect immunofluorescence staining of human endometrium with anti-dipeptidyl peptidase (DPP) IV monoclonal antibody, (a) Midproliferative phase; (b,e) mid-secretory phase; (c,f) late secretory phase; (d) the first trimester of pregnancy (6 weeks of gestation). DPP IV expression on endometrial glandular cells (GL) was weak in proliferative phase (a) whereas the expression was strong in mid- (b) and late (c) secretory phase and during the first trimester of pregnancy (d). Endometrial surface epithelium (SE) also showed strong expression of DPP IV in the mid-secretory phase (e). Endometrial stromal cells (ESC) and decidual cells (DC) did not express DPP IV ( a - f ) . Apical membrane of GL and SE appeared to express DPP IV stronger than the basolateral membranes (b-f). Heterogeneity of DPP IV expression in GL was shown in (f). a-f: Magnification X140.
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Fresh tissue samples were cut into small pieces, embedded in OCT compound (Miles Inc., Elkhart, IN, USA), frozen with Liquid nitrogen, and kept at -80°C until sectioned. Cryostat (Reichert-Jung, Heidelberg, FRG) sections, ~ 6 /tm thick, were air-dried and fixed with acetone at -20°C for 5 min. Non-
specific immunoglobulin binding was blocked by preincubation with 5% normal rabbit serum in phosphate-buffered saline (PBS, pH 7.4) for 15 min. The sections were then incubated for 1 h with the monoclonal antibody in a moist chamber at room temperature. After three washes with PBS, they were incubated for 30 min with FITC-conjugated rabbit anti-mouse immunoglobulin. After another three washes with PBS, the sections were examined with a fluorescence microscope (Nikon, Tokyo, Japan). For negative controls, the first antibody was replaced by normal mouse serum at an appropriate dilution. One cryostat section of each sample was stained with
Dipeptidyl peptidase IV In the human endometrium
haematoxylin and eosin, and was examined morphologically with a light microscope.
Results Indirect immunoflwrescence staining Endometria were obtained from 23 women, 35 to 48 years old, with regular menstrual cycles and deciduae were obtained from 11 women, 18 to 40 years old, at 6 —13 weeks of gestation. Ten endometria were in proliferative phase, four were in early secretory phase, four were in mid-secretory phase, and five were in late secretory phase. As shown in Figure 1 and Table I, DPP IV was expressed on endometrial glandular cells and surface epithelium. Judging from the fluorescence intensity, the expression of DPP IV on both these areas appeared weak during the proliferative phase (Figure la). The staining of DPP IV was stronger in the early secretory phase than in the proliferative phase (data not shown). In the mid- to late secretory phase, and in the first trimester of pregnancy, DPP IV expression was strong (Figure l b - e ) . It appeared that DPP IV expression on the apical membrane of glandular cells and surface epithelium was stronger than that on the basolateral membrane (Figure l b - f ) . Glandular cells in the basal layer of the endometrium also appeared to show the cyclic change of the DPP IV expression, although the expression was weaker than that in the functional layer (data not shown). In three
Histochemistry The endometria of 28 women, 37 and 49 years old, and nine first trimester deciduae, at 6 — 13 weeks gestation, were examined. Of the 28 endometria, 10 were in the proliferative phase, five were in early secretory phase, six were in midsecretory phase, and seven were in late secretory phase (Table H). DPP IV enzyme activity was rarely detected in endometrial glandular cells and surface epithelium in the proliferative and early secretory phases (Figure 2a), but in the mid-secretory phase, DPP IV activity became detectable (Figure 2b). In the late secretory phase, glandular cells and surface epithelium had strong DPP IV activity (Figure 2c). During the first trimester of pregnancy, DPP IV activity in both areas was slightly weaker than that in the late secretory phase (Figure 2d). There appeared to be no difference of DPP IV activity between glandular cells and surface epithelium (Figure 2b). The apical membranes of glandular cells and of surface epithelium appeared to show stronger DPP IV activity than the basolateral membranes (Figure 2c). Glandular cells in the basal layer had DPP IV enzyme activity in the mid-secretory phase and thereafter, but the activity appeared weaker than that in glandular cells of the functional layer (data not shown). In some samples in mid- and late secretory phases, DPP IV enzyme activity in the glandular cells was heterogeneous (Figure 2b). By contrast, endometrial stromal and decidual cells and other cells in the endometrial stroma did not have DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy, in any of the specimens which we have examined (Figure 2a -d). Although the endothelium of some vessels in the uterine muscle layer showed weak DPP IV activity, the endothelium of the vessels in the endometrium did not show any detectable DPP IV activity (data not shown). Uterine muscle did not show DPP IV enzyme activity (data not shown).
Table I. Immunohistological detection of dipeptidyl peptidase (DPP) IV Proliferative phase [n = 10) GL SE ESC/DC
Secretory phase Early (n = 4)
Mid (n = 4)
Late (n = 5)
First trimester of pregnancy (n = 11)
+- ++
+
+++
+H
+
h++
h+ +
GL = endometrial glandular cells; SE = endometrial surface epithelium, ESC = endometrial stromal cells; DC = decidual cells. Immunofluorescence intensity was arbitrarily graded as: — , negative; + weakly positive; + + , moderately positive; and + + + , strongly positive.
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Histochemical staining Unfixed cryostat sections, ~ 6 /on thick, were air dried, and kept at —80cC until assay, which was performed within 2 weeks. The enzyme-histochemical reaction for DPP IV was carried out according to the method of Lojda (1979), with minor modifications. Briefly, 4 mg of glycyl-prolyl-4-methoxy-/?naphthylamide (Bachem, Bubendorf, Switzerland) was dissolved in 0.5 ml N,N-dimethylformamide (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and mixed with 10 mg Fast Blue B salt (Sigma Chemical Company, St Louis, MO, USA) dissolved in 10 ml 0.1 M phosphate buffer, pH 7.3. The mixture was filtered through a 0.22 y.m membrane filter and immediately overlaid on slides. Incubation was carried out at room temperature for 6—7 min. After incubation, the sections were washed in distilled water, air dried, and observed with a light microscope. Glycyl-prolyl-4-methoxy-/3-naphthylamide has been reported to be a specific substrate for DPP IV in histochemistry (Lodja, 1979). As negative controls, cryostat sections were incubated with substrate-free media (Hartel etai, 1988).
out of nine mid- and late secretory phase endometrial samples, the grade of DPP IV expression on the glandular cells differed greatly from gland to gland in the functional layer, despite there being no obvious microscopic morphological differences between the glands (Figure If)By contrast, endometrial stromal and decidual cells, and other cells in the endometrial stroma, did not express DPP IV at any time throughout the menstrual cycle or during the first trimester of pregnancy (Figure l a - f ) . Although the endothelium of some vessels in the uterine muscle layer expressed DPP IV, the endothelium of vessels in the endometrium did not appear to express DPP IV (data not shown). Uterine muscle did not express DPP IV (data not shown).
K.Imai etal.
Table U. Enzyme-histochemical activity of dipeptidyl peptidase (DPP) IV ProUferative phase (n -10)
Secretory phase Early (n = 5)
Mid (n = 6)
+ ~+-f
GL SE ESC/DC
Late (n = 7)
:r
First trimester of pregnancy (n — 9)
I- + +
SE
GL
Fig. 2. Enzyme histochemistry showing dipeptidyl peptidase (DPP) IV activity in the human endometrium. (a) Proliferative phase; (b) midsecretory phase, (c) late secretory phase; (d) during the first trimester of pregnancy (8 weeks of gestation). DPP IV activity on endometrial glandular cells (GL) was rarely detected in the proliferative phase endometrium (a). DPP IV activity in GL and endometrial surface epithelium (SE) became detectable in the mid-secretory phase (b), and was strong in the late secretory phase (c) and during the first trimester of pregnancy (d). Endometrial stromal cells (ESQ and decidual cells (DQ did not express DPP IV activity during the menstrual cycle or the first trimester of pregnancy (a—d). DPP IV activity appeared stronger on the apical membrane than on the basolateral membranes of GL and SE (c). a,b and d; Magnification X70; c: magnification x 140.
Discussion The results presented in this paper can be summarized as follows. Firstly, on indirect immunofluorescence staining, both endometrial glandular cells and endometrial surface epithelium, but not endometrial stromal cells or decidual cells, express DPP FV/CD26 antigen throughout the menstrual cycle and during the first trimester of pregnancy. The immunoreactivity is weak in the proliferative phase, gets stronger in the early secretory phase, and remains strong in the mid- to late secretory phase, and in the first trimester of pregnancy. Secondly, histochemically, DPP IV enzyme activity is detected in endometrial glandular cells and 1192
endometrial surface epithelium in the mid-secretory phase and thereafter. Endometrial stromal cells and decidual cells show no detectable DPP IV enzyme activity. These findings are in parallel with those obtained by indirect immunofluorescence staining. Thirdly, the immunoreactivity and enzyme activity of DPP IV is stronger on the apical membrane of the endometrial glandular cells and endometrial surface epithelium than on the basolateral membranes. These findings indicate firstly that DPP IV is an excellent cell marker of endometrial glandular cells and endometrial surface epithelium; secondly that DPP IV can be considered to be a
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GL = endometrial glandular cells; SE = endometrial surface epithelium; ESC = endometrial stromal cells; DC = decidual cells. •Weakly positive GL were also sporadically noted in two out of 10 specimens. The staining was arbitrarily graded as: —, negative; + , weakly positive; + + , moderately positive; + + + , strongly positive.
Dipeptidyl peptidase IV in the human endometriom
antigen was shown to be identical to DPP IV (Mattern et al., 1989; Stein etal., 1989; Ulmer etal., 1990). This antigen is expressed strongly on activated T lymphocytes and on some resting T lymphocytes (Fox et al., 1984). Inhibition of DPP IV enzyme activity is shown to suppress T lymphocyte function (Schon et al, 1989; Flentke et al., 1991). Therefore DPP IV is considered to play an important role(s) in the immune system. Similarly, DPP IV on the endometrial glandular cells and on the endometrial surface epithelium might play some immunological role(s). Furthermore, DPP IV has been shown to be involved in cellular adhesion in extracellular matrix proteins (Hanski et al., 1985, 1988; Piazza et al., 1989; Dang et al., 1990). Since DPP IV is expressed on the apical membrane of endometrial surface epithelium during the peri-implantation period, this antigen might affect the attachment of fertilized eggs on to the endometrial surface. In this regard, Denker (1977) previously suggested the importance of proteinases in implantation. In conclusion, DPP IV is established as a differentiation marker for human endometrial glandular cells and endometrial surface epithelium. The role(s) of DPP IV in human reproduction remains to be clarified.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (no. 03454396) from the Ministry of Education, Science and Culture of Japan and by the Shimizu Foundation Research Grant for 1990.
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differentiation antigen for endometrial glandular cells and endometrial surface epithelium, since immunoreactivity and enzyme activity due to DPP IV are dependent on the stages of endometrial differentiation; and thirdly that biologically active DPP IV is present on endometrial glandular cells and on endometrial surface epithelium during the peri-implantation period and thereafter. It is well established that lymphocytes, granulocytes and macrophages are distributed in the human endometrium and decidua (Buhner and Johnson, 1985; Kamat and Isaacson, 1987), and DPP IV is shown to be present in some T lymphocytes (Lojda, 1977; Fox et al., 1984; Schon et al, 1984). However, our results showed that blood cells in the endometrium as well as endometrial stromal and decidual cells had no detectable DPP IV immunoreactivity or enzyme activity. Lojda (1979) reported that, histochemically, DPP IV activity in the endothelium of blood vessels was heterogeneous. In our study most of the endothelium in the uterine muscle layer was DPP IV-positive, but the endothelium in the endometrium did not show DPP IV, either histochemically or immunohistologically. This finding is consistent with Lojda's observation. DPP IV has been extensively studied both biochemically and histochemically, in various animals and in humans (Hopsu-Havu and Glenner, 1966; Gossrau, 1979; Heyman and Mentlein, 1984; Hartel et al., 1988; Heike et al., 1988). The presence of DPP IV in endometrial glandular cells and in the endometrial surface epithelium has been reported in the rat (Gossrau, 1979) and rabbit (Classen-Linke et al, 1987). Classen-Linke et al. (1987) reported changes in DPP IV activity on the endometrial epithelium during the peri-implantation period in a study using pseudopregnant and pregnant rabbits, and suggested that the changes might be due to steroid hormones or other mediators that may be produced by blastocysts. DPP IV is a membrane-bound ecto-enzyme, which exposes its catalytic site on the cell surface and removes mainly Xaa-Pro dipeptides from the N-termini of polypeptides (Yoshimoto et al., 1978; Heyman and Mentlein, 1984; Nausch et al., 1990). Its possible substrates include human gastrin-releasing peptide, human pancreatic polypeptide (Nausch et al., 1990), substance P (Puschel etal., 1982), human growth hormone-releasing hormone (Frohmann et al., 1989), and others. DPP IV can also degrade the alpha chain of human chorionic gonadotrophin (Nausch et al., 1990), which is secreted by trophoblasts. This suggests that DPP IV could affect the local metabolism of biologically active peptides present extracellularly in the female genital tract. Kenny et al. (1989) suggested the possibility that cell surface peptidase may have a key role in the control of growth and differentiation of many cellular systems in vivo by modulating the activity of peptide factors and regulating their access to adjacent cells. We have shown that the expression of aminopeptidase N, neutral endopeptidase, and DPP IV in the human endometrium is menstrual cycle dependent. Therefore, our present findings provide supporting evidence for Kenny et al.'s proposal (1989). DPP IV is also known to be a T lymphocyte surface antigen. Fox et al. (1984) reported Tal as a novel T lymphocyte-activation antigen, and it has been classified as CD26 antigen. Later CD26
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