Lectin binding sites on human endocervixA comparison with secretory and proliferative endometrium.код для вставкиСкачать
THE ANATOMICAL RECORD 215:262-266 (1986) Lectin Binding Sites on Human Endocervix: A Comparison With Secretory and Proliferative Endometrium YUN YEN, MAO-CHI LEE, MARTINA SALZMANN, AND N A N DAMJANOV Department of Pathology and Laboratory Medicine, Hahnemann University School of Medicine, Philadelphia, PA 19102-1192 ABSTRACT Endocervix and corresponding endometrium of women of reproductive age were studied histochemically with 13 fluorescein isothiocyanate-labeled lectins to delineate the differences between the epithelial cells in two anatomical sites. Lectin from Maclura pornifera (MPA), Ulex europaeus (UEA-I), Glycine max (SBA), and Vicia villosa (VVA) bound only to endocervical epithelium and were the only four lectins that distinguished endocervical from endometrial epithelium. These differences were independent of menstrual cyclic changes and blood group antigen secretion. These data show that lectins can be used to histochemically distinguish endocervical from endometrial glands. In our previous work Gee and Damjanov, 1985) we have shown that fluorescein isothiocyanate-labeled lectins can be used as histochemical reagents to specify pregnancy-associated changes in the uterus. Several lectins reacted differentially with pregnant and nonpregnant endometrial cells and were useful for histochemical distinction of pregnant and nonpregnant endometrium. Similar differential binding of lectins was found in the pregnant and nonpregnant mouse endometrium and oviduct (Lee et al., 1983). In the present study we have extended these lectin histochemical investigations to include human endocervix. We have compared lectin binding to endocervical and endometrial glands and show that some lectins can be used to distinguish cells in these two anatomical locations. We also show that lectin binding sites expressed during the proliferative phase of the menstrual cycIe remain on both endocervical and endometrial glands in the secretory phase and that the lectin binding to these cells does not depend on the blood group antigen secretion. MATERIALS AND METHODS from these uteri were taken from the fundus, body, and isthmus to determine whether there are any differences between various sites. Since uterine glands express blood group antigens in secretors (Szulman, 1960, 19621, the secretor status of individuals was determined by indirect immunofluorescence on parafin sections of endometrial tissue using monoclonal antibodies to these antigens (Dako, Santa Barbara, CA). Specimens showing blood group reactivity in blood vessels but not in glands were considered to be from nonsecretors, and those showing reactivity of both glands and vessels were secretors. There were 8 specimens from blood group A, 3 from blood group B, and 5 from blood group 0 individuals; 7 were from secretors and 9 from nonsecretors. These data corresponded to the serologically determined blood groups of these patients. All tissues were fixed in 4% formaldehyde solution €or 12 h r and embedded in parafin. Paraffh sections (5 pm) were prepared routinely and deparaffinized in xylene and rehydrated in graded ethanol. Following a brief rinse in distilled water, the sections were treated with 0.4% pepsin in 0.01 N hydrochloric acid for 2 hr at 37°C (Ekblom et al., 1982) and washed twice with phosphate-buffered saline (PBS). Sections were then incubated with various FITC-labeled lectins, listed in Table 1, in a moist chamber a t room temperature for 30 min. Before incubation, FITC-labeled lectins were diluted in PBS to a concentration of 50 pg/ml. A11 FITC-labeled lectins and sugars were purchased from E.Y. Laboratory (San Mateo, CA). The con- Endometrial and endocervical tissues from women of reproductive age were obtained by curettage or hysterectomy. The phase of the endometrial cycle was determined by the last menstrual period and was corroborated by histology by means of standard histologic criteria for dating of human endometrium. Fourteen specimens were collected by curettage: Seven specimens were collected during the midproliferative phase (days 7-10), and seven specimens during the secretory phase. Two Fig. 1. Tissues reacted with FITC-lectin from Maclura pornifera specimens corresponded to day 16, two to day 20, two to day 22-23 and one to day 26-27 of a 28-day cycle. Each (MPA). a. Endocervix, secretory phase, shows strong cytoplasmic reacin most cells. Some cells are nonreactive (arrows) ~ 2 2 0 .b. specimen was composed of endocervical and endometrial tivity Endometrium in secretory phase is nonreactive x 220. tissues. Two additional hysterectomy specimens (one in proliferative and one in day 18 secretory phase) removed for leiomyoma were systematically examined. Samples Received August 19, 1985; accepted February 19, 1986. 0 1986 ALAN R. LISS, INC. 263 LECTIN BINDING TO ENDOCERVIX TABLE 1. Lectins used and corresponding inhibiting sugars Carbohydrate specificities Source Lectin SUC. ConA WGA STA SBA VVA PNA GSA-I MPA RCA-I RCA-I1 UEA-I PHA-E PHA-L Canaualia ensiformis Triticum uulgare Solanum tuberosum Glycine max Vicia uillosa Arachis hypogaea Griffonia simplicifolia Maclura pomifera Ricinus communis Ricinus communis Ulex eurouaeus Phaseolug uulgaris Phaseolus uulgaris Inhibiting sugar a-D-Man> a-D-Glc (@-(1 +~)-D-G~CNAC)~, sialic acid (P-(1+4)-D-GlcNAc)' a-D-GalNAc=P-D-GalNAc a-D-GalNAc 0-D-Gal-(1- 3)-D-GalNAc a-D-Gal a-D-GalNAc> D-Gal 6-D-Gal> a-D-Gal a- or 0-D-Gal=D-GalNAc a-L-Fuc Complex Complex D-GlcNAc = N-acetyl-D-glucosamine;D-GalNAc = Methyl a-mannopyranoside D-GlcNAc D-GIcNAc D-GalNAc D-GalNAc D-Gal D-Gal D-GalNAc D-Gal D-Gal L-FUC D-GalNAc D-GalNAc N-acetyl-D-galactosamine; D-Gal = D-galactose; L-Fuc = L-fucose. TABLE 2. Lectin binding sites on human endometrium and endocewix' Lectin SUC. ConA WGA STA SBA VVA PNA GSA-I MPA RCA-I RCA-I1 UEA-I PHA-E PHA-L Proliferative phase Endometrium Endocervix gland epithelium gland epithelium ++ ++ ++ - +or - + ++ + -' + ++ + + + +-+ ++ + ++ ++ + + Secretory phase Endometrium Endocervix gland epithelium gland epithelium ++ ++ ++ - + or- -' - + +- + or -' + or -' + ++ + + + ++ ++ + ++ ++ + + 'Symbols: The intensity of Lectin binding was subjectively marked as negative (-), faintly visible (+), weak (+), and strong (+ +). 2PNA stains some glands but not others in both the secretory-phase and proliferative-phase endometrium. PHA-E and PHA-L stain some glands but not others in the secretory endometrium. 264 Y.YEN, M.-C. LEE, M. SALZMANN, AND I. DAMJANOV RESULTS The findings are summarized in Table 2 and illustrated in part in Figures 1-4. Endocervix All 13 lectins used in this study, except GSA-I, reacted with endocervical epithelium (Figs. 1-4). The reactivities of the lectins MPA (from Maclura pornifera) (Fig. 1) and UEA-I (from Ulex europaeus)(Fig. 2 ) were estimated as strong, whereas SBA (from Glycine rnax) (Fig. 3) and VVA (from Vicia uillosa) (Fig. 4) reacted less intensely. These lectins reacted with most but not all endocervical cells. The unreactive cells were interspersed individually with reactive cells (Figs. l a , 2b) or formed groups covering short segments of the mucosal surface (Fig. 3). In some areas essentially all cells reacted with the lectins (Fig. 2a). In most instances the cytoplasmic lectin binding sites were both apical and basal, although in some instances the apical reactivity appeared more intense (Fig. 4). Mucus within the lumen of some glands reacted also with all four lectins (Figs. l a , 3). Endometrium Epithelium of endometrial glands in proliferative phase reacted with 8 lectins: SUC.ConA, WGA, STA, PNA, RCA-I, RCA-11, PHA-E, PHA-L. The corresponding luminal epithelium reacted with all those lectins. SBA reacted with luminal epithelial cells but was unreactive with glandular cells, as reported previously (Lee and Damjanov, 1985). No difference was noted in the reactivity of glands in the basalis and functionalis. Tissues sampled from fundus, body, and isthmus all reacted in a n identical manner, Epithelium obtained from secretory endometrium showed the same lectin binding pattern as the proliferative phase samples (Figs. 1,2). Comparison of Endometrial and Endocervical Epithelium Three lectins (MPA, UEA-I, and VVA) reacted exclusively with endocervical glands (Figs. 1-4). SBA reacted with endocervical glands and luminal epithelium of the endometrium but was unreactive with endometrial glands. The blood group and the secretor status of individual tissue donors did not influence the reactivity of either endocervical or endometrial epithelium. DISCUSSION Fig. 2. Tissues reacted with FITC-lectin from Ulex europaeus WEAI). a. Endocervical glands in secretory phase show strong reactivity uniformly in all cells. x350. b. Another area of cervical glandular epithelium from the same specimen shows uneven reactivity of cells, accounting for the so-called mosaic pattern. X 350. c. Endometrial glands, secretory phase, are unreactive. x220. trol sections were incubated in a solution containing FITC-labeled lectins and 0.2 mmoles of the specific inhibitory sugar(s) listed in Table 1.Following incubation, the slides were washed in PBS, coverslipped with glycerin, and examined with a U V microscope. The present study shows that 12 lectins of different carbohydrate specificities react with endocervical glands. However, all except four reacted also with endometrial glands, as reported previously (Lee and Damjanov, 1985). Since MPA, UEA-I, SBA, and W A show differential reactivity, these four lectins could be used as markers to distinguish endometrial from endocervical glands. Three cell types were identified in the endocervix by electron microscopy: mucus cells, cells similar to the epithelial cells of the endometrium, and ciliated cells (Nilsson and Westman, 1961). Since mucus-secreting cells of endocervix are different from either luminal or glandular epithelial cells in the endometrium, most of the differential binding of the four lectins could be due to their reactivity with endocervical mucus-secreting cells. These cells form the majority of endocervical cells. The endocervical cells that are unreactive with lectins LECTIN BINDING TO ENDOCERVIX 265 Fig. 3. Tissues reacted with FITC-lectin from Glycine mat: (SBA) show reactivity of secretory phase endocervical epithelium in one area and no reactivity in the adjacent segment (arrows). Intraluminal mucus is strongly reactive. ~ 2 2 0 . Fig. 4. Tissue reacted with FITC-lectin from Vicia uillosa W A ) . In the endocervical glandular epithelium in secretory phase, cells of strong reactivity are intermixed with nonreactive cells. ~ 2 2 0 . probably correspond to ciliated cells or to cells similar to epithelial cells of the uterine body (Nilsson and Westman, 1961). The endocervical glands undergo cyclic morphological changes (Bradburn and Webb, 1951). The physical properties of endocervical mucus also change during the menstrual cycle (Odeblad, 1962, 1968; Davajan and Kunitake, 1969). However, there is no evidence that the biochemical composition of the mucus undergoes cyclic changes (Schumacher, 1970). Our findings disclose no menstrual period-related differences in the lectin binding to intracellular or extracellular mucin and are thus consistent with previous studies indicating that the biochemical composition of mucus does not change during the menstrual cycle. Odeblad (1969) and Schumacher and Pearl (1969) have shown qualitative differences in the secretion of different glands from different portions of the endocervix that account for the so-called “mosaic pattern” of epithelial secretions. The present lectin histochemical study clearly outlines this mosaic pattern within individual glands as well as within groups of glands. Thus lectins are a good probe for studying the heterogeneity of cell populations as well as glands in the cervix. Szulman (1960,1962) showed blood group antigens on the endocervical and endometrial glands of secretors. We have examined tissues from both secretor and nonsecretor donors and see no difference in lectin binding to their endocervical glands. Thus, the reactivity of these 12 lectins to endocervical glands does not depend on the secretor status or the blood group of the donor. In summary, the present study shows that FITC-lectins can be used to distinguish normal endocervical from endometrial glands. These changes probably reflect the histological and functional differences between these two anatomically and developmentally related epithelia. Next, we plan to use these histochemical data in the study of uterine tumors in order to distinguish endometrial from endocervical neoplasms. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants CA-23097 and CA-38405 and a fellowship from the Lalor Foundation, Wilmington, Delaware. The skillful secretarial help of Ms. Jacklyn Powell is gratefully acknowledged. LITERATURE CITED Bradburn, G.B., and C.F. Webb (1951) Cyclic variations in the endocervix. Am. J. Obstet. Gynecol., 62997-1008. Davajan, V., and G. Kunitake (1969) Fractional in vivo and in vitro examination of postcoital cervical mucus in the human. Fertil. Steril., 20: 197-210. Ekblom, P., M. Miettinen, J. Rapola, and J.M. Foidart (1982) Demonstration of laminin, a basement membrane glycoprotein in routinely processed formalin fixed human tissues. 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Schumacher, G.F.B., and M.J. Pearl (1969) Cyclic changes of muramidase (lysozyme) in cervical mucus. J. Reprod. Med., 3~171-178. Seulman, A.E. (1960) The histological distribution of blood group substances A and B in man. J. Exp. Med., 111:785-799. Szulman, A.E. (1962) The histological distribution of the blood group substances in man as disclosed by immunofluorescence. II. The H antigen and its relation to A and B antigen. J. Exp. Med., 115:977995.