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Differentiation of human trophoblast populations involves alterations in cytokeratin patterns.

J Mhlhauser, C Crescimanno, M Kasper, D Zaccheo and M Castellucci J Histochem Cytochem 1995 43: 579 DOI: 10.1177/43.6.7539466 The online version of this article can be found at: http://jhc.sagepub.com/content/43/6/579

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The Journal of Histochemistry and Cytochemistry Copyright 0 1995 by The Histochemical Society, Inc.

Vol. 43, No. 6. pp. 579-189. 1995 Printed in U.S.A.

Original Article

1
Differentiation of Human Trophoblast Populations Involves Alterations in Cytokeratin Patterns
JUDITH MUHLHAUSER, CATERINA CRESCIMANNO, MICHAEL KASPER, DAMIANO ZACCHEO, and MARIO CASTELLUCCI
Laboratory of Vascular Pathology, Istituto Dermopatico delllmmacolata, 101, Rome, Italy (JM); Institute of Anatomy, University of Genova, Genova, Italy (CC,DZ); Institute of Pathology, Medizinische Akademie Carl Gustav Carus, Dresden, Gennany (MK); and Institute of Normal Human Morphology, University of Ancona, Ancona, Italy (MC).
Received for publication August 4, 1994 and in revised form January 31, 1995; accepted February 16, 1995 (4A3452)

Cytokeratins (CKs)are related to proliferation and differentiation of epithelial cells. Little knowledge exists about CK patterns in human trophoblast subpopulations (villousand extravilloustrophoblasts). To better understand differentiation and function of trophoblast components, we studied the distribution patterns of CKs in the placenta throughout pregnancy. A panel of well-defined monoclonal antibodies against different types of cytokeratins, vimentin, and fibrin, was used on frozen and paraffin sections. CK8, 18, and 19 were expressed in all the villous and extravilloustrophoblastic subsets throughout pregnancy. In the first trimester, syncytiotrophoblasts were positive for CK7 and 13 along the basal membrane. As pregnancy progressed there was an increase in intensity of the reaction product and a more diffuse positive staining of CK7 in the cytoplasm of the syncytium, with evident positivity along the apical membrane. CKU showed similar expression as CK7, but with less intense staining along the apical membrane and less prominent staining in the cytoplasm. Villous cytotrophoblastswere also positive for CK7 and CK13. CK17 was found related

to cytotrophoblastic cells in contact with or next to fibrin deposits. Extravillous cytotrophoblastsin cell islands and cell columns were positive for CK13 only in the cell layers located proximal to the villous stroma, whereas the distal and more differentiated cells were negative. CK7 was positive in all epithelial cells of cell islands and columns, but the reaction product was not present in cells deeply migrated into the decidua. Amnion was negative for anti-CK13 antibodies in the first trimester but was positive at term. CK4 and CKl6 were not found in the placenta. Our study shows for the fmt time that the different populations of human placental trophoblast express cytokeratins in developmental, differentiative, and functional specificpatterns. These findings can be useful to distinguish and classify the various trophoblastic populations and provide a foundation for studying pathological aspects of the trophoblast. (JHisrochem C y r h e m 43:577-589, 1995)
KEY WORDS: Cytokeratins; Human trophoblast; Human placenta;

Immunohistochemistry.

Introduction
The human placenta is characterized by a fine balance between proliferation, differentiation, and invasive processes of the trophoblast. The trophoblast may be considered as a true epithelium with most remarkable characteristics. During placental development,various subsets of trophoblasts originate from the trophectoderm of the blastocyst, including the villous trophoblast, which covers the placental villi, and the extravillous cytotrophoblast of cell islands and cell columns. The villous trophoblast consists of the highly differentiated syn-

Supported by a grant from the Consiglio Nationale della Ricerche (C.N.R.) 91.00269.PF41. Correspondence to: Judith Miihlhauser, Lab. Cardiovasc. Sci.. GRC, NIAINIH, 4940 Eastern Ave., 3D06, Baltimore, MD 21224.

cytiotrophoblast(surface layer) and the cytotrophoblast(basal layer) (Benirschke and Kaufmann, 1990). In first-trimesterplacental villi, syncytiotrophoblast and cytotrophoblast form a compact two-layered epithelium. As pregnancy progresses, fusion of cytotrophoblastic cells with the syncytiotrophoblast leads to a largely single-layered epithelium of syncytial character, with only occasionally underlying proliferative cytotrophoblastic cells. The syncytiotrophoblast is in contact with the trophoblast basement membrane not only in the second half of pregnancy but even in the first trimester, through thin cytoplasmic projections extending between the cytotrophoblasticcells (Benirschke and Kaufmann, 1990; Dearden and Ockleford, 1983). Therefore, according to Bosch et al. (1988), the villous trophoblast can be considered a simple epithelium. Cytotrophoblastic cell islands and cell columns exhibit a completely different architecture. Indeed, these two epithelial structures are composed of stratified/multilayered extravillouscytotrophoblastic cells. Cell columns are responsible for the attachment

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of placental villi to the basal plate and are interposed between fetal (villous) and maternal connective tissue (basal plate). Cell islands are comparable structures which are free-ending in the intervillous space. Extravillous cytotrophoblastic cells of cell islands and cell columns located proximal to the villous stroma have been identified as proliferating cells, whereas extravillous cytotrophoblastic cells located distal to the villous stroma show more differentiated characteristics and are no longer proliferative (Muhlhauser et al., 1993; Castellucci et al., 1991; Bulmer et al., 1988). The most distally located of these no longer proliferating epithelial cells are able to leave the cell columns and migrate into the decidua, becoming interposed (interstitial cells) between maternal decidual cells. Therefore, such extravillous cytotrophoblastic cells exhibit some pseudomalignant features with a highly regulated invasive capacity (Lala and Graham, 1990; Ohlsson, 1989; Fisher et al., 1985,1989). In recent years, normal epithelia and malignancies derived therefrom have been characterized and classified by their cytokeratins (CKs), a subclass of intermediate filaments (Moll et al., 1988; Sun et al., 1985; Ramaekers et al., 1983). Thus far, 20 CKs have been identified in human epithelial cells and catalogued by Moll et al. (1982a,1990) according to their isoelectric point and molecular weight. It has been shown that different CK polypeptides are expressed in a characteristic tissue-specific and differentiation-related manner (Moll et al., 1982a; Franke et al., 1981). For example, simple epithelia such as intestinal epithelia express CK8 and CK18, whereas the stratified epithelium of the esophagus predominantly expresses CK4 and CK13. Few data are available on the expression of CKs in the human placenta (Daya and Sabet, 1991; Khong et al., 1986; Clark and Damjanov, 1985), as well as in primate placenta (Blankenship and King, 1993; Blankenship et al., 1993). As considered above, the various trophoblastic subsets consist of different types of epithelia. Therefore, it is possible that proliferative and differentiative processes of villous and extravillous trophoblasts are accompanied by alterations in the CK patterns. Therefore, we used a panel of well-defined monoclonal antibodies (MAbs) to CK polypeptides to characterize the various trophoblast populations as well as the amnion epithelium throughout pregnancy. In addition, the alterations in the CK expression of the trophoblast in contact with deposits of fibrin were also studied. The latter is an additional extracellular matrix component that is present in the placenta throughout pregnancy (Benirschke and Kaufmann, 1990). We show that various trophoblast subsets express different types of CKs during gestation and that fibrin can locally interfere with CK expression. Our data are discussed in relationship to differentiation processes of the trophoblast populations.

Tissue Preparatio n for Immunohistochemistry

Placentas were cut into small blocks not exceeding 0.5 mm. Some tissue blocks were snap-frozen in liquid nitrogen and stored at 20C until use Sections ( 5 pm) were cut with a cryostat and air-dried. Before further procedures, frozen tissue sections were either fixed in Carnoys fixative (10 min at room temperature) or in acetone (10 min at 4C). or were used unfixed. Other tissue blocks were fixed for 1 hr at 4C in 4% buffered formalin or overnight in an acid-alcohol fixative solution (Battiford and Kopinski. 1986) and routinely processed for paraffin embedding at 56C as previously described (Miihlhauser et al., 1993). Paraffin sections (6 pm) weir cut and stretched at 4 5 T , allowed to dry, and stored at 4C until use.
-

Immunohistochemistry
Immunofluorescence. Fixed and unfixed frozen sections were incubated for 20 min at room temperature (RT) with 1.5% bovine serum albumin in PBS. Subsequently, sections were incubated with a primary antibody (Table 1) at appropriate dilutions for 1-2 hr at RT or overnight at 4C. Slides were washed twice in PBS and then incubated for 1 hr with the second antibody F 232 (FITC-conjugated rabbit anti-mouse; Dako; Carpinteria. CA) or F 205 (FIX-conjugated swine anti-rabbit; Dako). both diluted 1:20 in PBSlBSA 1.5%. After two washes in PBS, sections were mounted under coverslips with N-propyl gallate, prepared accordingly to Giloh and Sedat (1982).

Immunoperoxidase. Some fixed frozen sections as well as paraffin sections. the latter dewaxed and treated by protease (Pronase; Serva. Paramus, NJ) 0.1% in PBS for 13 min at 37C. were processed for immunoperoxidase techniques by using a rabbit anri-mouse streptavidin-biotin system (S-ABC; Zymed; South San Francisco, CA) or a goat anti-mouse streptavidin-biotin system (Vectastain Elite Kit; Vector Laboratories, Burlingame. CA) foilowing the kit instructions.
Controls. Negative and positive controls were performed. For negative controls. in all experiments the primary or the secondary antibodies were replaced by PBSiBSA 1.5%. No staining pattern was observed. For positive controls, the cytokeratin antibodies were applied to cryostat or paraffin sections of human tonsil, eye, urothelium. and esophagus. The antibodies to glial fibrillary acidic protein were used on paraffin and cryostat sections of the third ventricle of rat brain. All positive controls showed appropriate reaction patterns. In addition, MAbs LLOO1. LL002, SK2-27, and E3. as well as GF 12-24 and the GFAP polyclonal antibody, were also used to exclude possible crossreactivity of Ks 13.1 with CK14, CK17, and with the glial fibrillary acidic protein. as suggested by the manufacturer

Results
The immunohistochemical results are summarized in Table 2 (villous and extravillous trophoblast). Different antibodies against the same antigen provided identical results in the placental tissue used in this study. In addition, various methods of fixation and embedding revealed identical expression patterns. Therefore, no separate description will be provided.

Materials and Methods

Tissues
Twenty-two human placentas aged 7 ( n = 3). 9 ( n = 2 ) , 12 (n = 4), 14 ( n = 3), 18 ( n = 2), 33 (n = 4). and 40 (n = 4) weeks of gestation were collected from clinically normal pregnancies interrupted by curettage for psychosocial or medical reasons that were unlikely to affect placental structure and function, or terminated by cesarean sections and normal vaginal deliveries.

Villous Eopho blast

In the first trimester the syncytiotrophoblast was positive for CK7 mainly along its basal plasma membrane, whereas its apical plasma membrane was weakly fluorescent (Figure 1A). From the first to the second trimester the immunostaining for CK7 in the syncytium increased, and at term the syncytiotrophoblast was most heavily

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Table 1. Antibodies used in this study

Antibodya Biochemical specificity Dilution Source/Reference

MAb Lu 5 MAb 6B10 MAb 215B8 MAb Ks 7.18 MAb 4.1.18 MAb Ks 13.1 MAb 2D7 MAbs LLOOl and LL002 MAb SK2-27 MAb LMM3 MAb E3 MAb CY-90 MAb A>3-B/A2 GFAP PAb MAb GF 12-24 Vimentin MAb Fibrin MAb
a

CK1-19 CK4 CK4 CK7 CK8 CK13 CK13 CK14 CK14 + 17 CKl6 CK17 CK18 CK19 Glial filament protein Glial filament protein Vimentin Fibrin

Undiluted Undiluted 1:lO 1:lO 1:lO 1:2-1:lO 1:2-1:lO Undiluted 1:50 Undiluted Undiluted 1:1200 Undiluted 1:70 Undiluted Undiluted 1:lOO

Boehringer Mannheim (Milano, Italy) Van Muijen et al., 1986 Boehringer Mannheim Amersham (Arlington Heights, IL) Boehringer Mannheim Progen (Heidelberg, Germany) Euro-Diagnostic (Apledoorn, Netherlands) Purkis et al., 1990 Cintorino et al., 1988 Lane and Alexander, 1990 Progen Sigma Chemicals (St. Louis, MO) Karsten et al., 1985 Dako (Glostrup, Denmark) Progen Immunotech (Marseille, France) Immunotech

MAb. monoclonal antibody; PAb, polyclonal antibody

stained throughout the cytoplasm (Figure 1B). Villous cytotrophoblast showed strong positivity for CK7 throughout pregnancy (Figures 1A and 1B). The trophoblast of first-trimester placental villi was positive for CK13 (Figures 1 C and ID). The cytotrophoblastic cells appeared to contain a rim of intermediate filament-rich cytoplasm along the cell borders, whereas the basal part of these cells was negative (Figures ID, 2A, and 2B). In the syncytiotrophoblast, the apical part was very weakly stained or unstained for CK13, whereas the basal membrane was more strongly labeled (Figures 1C and 1D). CK13 immunoreactivity along the entire basal plasma membrane of the syncytiotrophoblast was more clearly recognizable where syncytiotrophoblast was in direct contact with the basement membrane (Figure ID) and in cell islands surrounded by syncytiotrophoblast (see Figure 6C), where the reaction product in the basal part of the syncytium was particularly evident owing to the absence of immunostaining in the underlying extravillous cytotrophoblast (see Figure 6C). In full-term placentas, the basal part of the syncytiotrophoblast was more strongly labeled than the apical part (Figure 3). Staining patterns in villous trophoblast, similar or comparable

to those revealed by Ks 13.1 antibody and 2D7 antibody, were not revealed with antibodies against antigens that might crossreact with Ks 13.1 (see Materials and Methods). With the polyclonal GFAP antibody, some fine staining was found in large vessels in the chorionic plate and around larger vessels of stem villi near the chorionic plate (data not shown). No immunostaining was observed with the MAb GF12-24 recognizing glial filament protein. MAbs LLOOl and LL002 (both specific for CK14) did not show reaction patterns. The MAbs E3 (anti-CK17) and SK2-27 (anti-CK14+17) showed specific staining at very localized positions (see below). Expression of CK17 was found in cytotrophoblastic cells associated or in contact with fibrin deposits in all placental samples used in this study (Figures 4A and 4B). The MAbs E3 and SK2-27, recognizing CK17 and CK14 + 17, respectively, showed identical staining patterns, whereas no staining was observed in the placenta with anti-CK14 antibodies. Sporadically,single cytotrophoblast cells apparently not in contact with fibrin were also positive for CK17. Term placental villi completely surrounded by fibrin showed the largest number of trophoblast cells positive for CK17 (Figures 5A and 5B).

Table 2. Immunohistochemical results for villous trophoblasts ana' extravillous trophoblasts

Histological location" Gestational age Cytokeratios expressed

Syncytiotrophoblast

First half of pregnancy

Second half of pregnancy Cytotrophoblast Proximal extravillous cytotrophoblast in CI and CC Distal extravillous cytotrophoblast in CI and CC Extravillous trophoblast migrated into BP
CI, cell islands; CC. cell columns; BP, basal plate

First and second halves of pregnancy First and second halves of pregnancy First and second halves of pregnancy First and second halves of pregnancy

7 strong basal cytoplasm, weak apically, 13 basal cytoplasm, 8. 18, 19 7, 8, 18, 19, 13 strong basal and weaker apical cytoplasm 7, 8, 13, 18, 19, 17 associated with fibrin deposits 7. 8. 13, 18, 19 7. 8, 18, 19 8, 18, 19

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MUHLHAUSER.CRESCIMANNO, KASPER, ZACCHEO. CASTELLUCCI

i'
Figure 1. Immunofluorescence staining of frozen sections incubated with MAbs (A,B) Ks 7.18 (anti-CK7) and (C,D) Ks 13.1 (anti-CK13).CKbpositive staining patterns are found in villous cytotrophoblast and weakly along the apical plasma membrane of the syncytiotrophoblast in the first trimester of gestation (A), whereas MAb Ks 13.1 (anti-CK13)shows a specific reaction in the cytotrophoblast and a less intense the cytoplasm of the syncytiotrophoblast is heavily stained at term (E). reaction along the apical plasma membrane of the syncytiotrophoblast in first trimester placenta (C). Higher magnification of an area depicted in C is shown in D. A rim of reaction pattern for CK13 is found in the cytotrophoblast cells, but the basesof the cells are negative (arrow). The apical syncytium is slightly immunoreactive. The basal part of the syncytium overlying the cytotrophoblast and contacting the basal lamina (arrowhead) ISpositive. vs. villous stroma. Bars = 20 rim.

Trophoblasts of human placental villi from first, second, and third trimesters were strongly immunoreactive with antibodies against pan-cytokeratin, CK8, 18. and 19. In full-term placentas the media of larger vessels also showed positive staining for CK8 and CK18. Expression patterns for CK4 and CKl6 were not found at any stage of gestation in the human placenta.

Extravillous Fop hoblast

In cell islands and cell columns, extravillous cytotrophoblasts were positive for pan-cytokeratin, CK7 (Figure 6 A ) , CK8, CK18, and

CKI9 (Figure 6B). The distribution of CK13 was peculiar in these epithelial structures. CK13 showed staining in cells located proximal to the villous stroma, whereas with increasing distance from the villous stroma this polypeptide was no longer expressed. Distally located extravillous trophoblastic cells were negative for the Ks 13.1 antibody (CK13) (Figure 6C). In the basal plate. cytotrophoblastic cells were strongly positive for pan-cytokeratin, CK8. CK18. and CK19 (Figure 7A). Different staining patterns, depending on the location of the cells in the basal plate, were found for CK7. Extravillous cytotrophoblasts that just invaded the basal plate showed high expression of CK7. but with

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increasing distance from the anchoring villi (cytotrophoblastic cell columns) the immunostaining ceased, leading to completely negative cells in profound areas of the basal plate (Figure 7B). CK13positive extravillous cytotrophoblastic cells were detected only at the attachment site of the anchoring villi. Extravillous cytotrophoblastic cells found throughout the basal plate were minimally or not at all stained (Figure 7C). Decidual cells stained positive for vimentin. but not for cvtokeratin antibodies.

Amnion

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CK13 was not detectable in first-trimester amniotic membrane, but it was expressed in the amnion of term placenta (Figures 8A and 88). Our investigation confirms the results of Regauer et al. (1985) for other CK types.

Discussion
The human placenta is a highly complex organ, which undergoes continuous remodeling of its epithelial components (villous and extravillous trophoblasts) throughout pregnancy (Benirschke and Kaufmann, 1990; Castellucci et al., 1990). Our study shows for the first time that temporal, differentiative, and structural changes of the trophoblast subsets during gestation are characterized by and related to specific patterns of intermediate filaments. The most interesting differences relate to cytokeratins 7, 13, and 17. CK7 is frequently represented in normal simple epithelia (Kasper et al., 1991; Ramaekers et al.. 1987). In villous trophoblast, which may be considered as a "simple epithelium" (see Introduction), we have shown that CK7 and CK13 expressed in the villous trophoblast undergo an increase in reactivity and redistribution into the syncytium during gestation. Our immunohistochemical findings of CK13 in the villous trophoblast (simple epithelium) are remarkable, considering that CK13 is usually absent in simple epithelia but is present in most internal stratified epithelia as well as in the transitional epithelium (Kuruc et al.. 1989; van Muijen et al., 1986; Nagle et al.. 1985; Sun et al., 1985). Our data are supported by previous CK13 mRNA detection in human placenta (Kuruc et al.. 1989). CK17 is not commonly found in human epithelia. Its expression in normal tissue has been shown in only a few simple and stratified internal epithelia (Kasper et al., 1991; Guelstein et al., 1988; Moll et al., 1982b). Therefore, the expression of CK17 in the cytotrophoblast in contact with perivillous fibrin deposits is an important result of our study. Such deposits are a common histological feature in normal and pathological placentas during gestation (Nelson et al., 1990; Fox, 1978). As has been previously pointed out (Nelson et al., 1990). these limited deposits of fibrin are believed to be a form of villous repair and they frequently show partial or complete epithelialization by the trophoblast. The latter is in direct contact with fibrin, and it is assumed that trophoblastlfibrin interactions could also modulate trophoblastic differentiation et '99'). Some studies have pointed Out that epithelial cell-extracellular matrix interactions, as well as the composition of the extracellular matrix and the different amounts of its various components,can have 'pecific influencein determining differentiated keratin protein expression (Kurpakus et al.. 1992; Mackay et al., 1990; Kolega et al., 1989). Our study shows for the

Figure 2. Two parallel frozen sections of first trimester placental villi, tangentially sectioned. (A) Toluidineblue section. (E) Immunofluorescence.MAb 2D7 (anti-CK13)-positive reaction pattern is continuous around the entire cytotrophoblastic cell. vs. villous stroma. Bars = 20 vm. Figure 3. Full term placental villi showing immunofluorescence staining patterns for CK13 (using MAb Ks 13.1) along the basal and apical plasma membrane of the syncytiotrophoblast. Cryostat section. VS. villous stroma. Bar = 20 pm.

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Figure 4. Two parallel frozen sections of a first-trimester placental villus. Immunofluorescencestaining patterns with antibodies (A) E 3 (anti-CK17) and (B) antifibrin. Note that corresponding areas are positive. vs. villous stroma. Bar = 20 um.

first time that the expression of CK17 is associated with and in close proximity to fibrin deposition in human placenta. Further studies could ascertain if fibrin may induce CK17 in the cytotrophoblast. The functional meaning of this limited CK17 expression in villous cytotrophoblastic cells could be its involvement in repair of damaged villous surfaces, probably by enhancing specific activities of cytotrophoblastic cells. Other extracellular matrix molecules, especially basement membrane components, have been shown to have specific influence in triggering the production of certain keratins in epithelial cells (Kurpakus et al., 1992; Mackay et al., 1990; Kolega et al., 1989). In cytotrophoblastic cell islands and cell columns, basement membrane molecules have recently been found to be expressed in a nonpolarized fashion, i.e., around and between the cytotrophoblastic cells (Castellucci et al., 1993; Damsky et al., 1992; Autio-Harmainen et al.. 1991). In addition, recent studies (Castellucci et al., 1993; Damsky et al., 1992) have pointed out that different basement membrane molecules, such as laminin, collagen IV, and fibronectin. show extensive modulation of their expression in cell islands and cell columns, i.e., most of these molecules are strongly expressed in the distal part of these epithelial structures. These data. together with the fact that different types of integrins are found at different lev-

els of the cytotrophoblastic cell columns (Damsky et al., 1992)probably playing a role in signal transduction from the extracellular matrix to the epithelial cells (see Werb et al., 1989)-couId be one explanation for the non-typical CK expression for stratified/multilayered epithelia in cell islands and cell columns. Indeed, CK8 and CKl8 present in all the cells of these two epithelial structures are typical CKs found in simple epithelia (Moll et al.. 1982a). Alterations of CK7 and CK13 expression patterns in the extravillous trophoblast are one of the most striking features of this study. CK7 is present in all the epithelial cells of cell islands and cell columns, but not in those leaving the cell columns and migrating deep into the decidua. CK7 has not been found previously in stratified/multilayered epithelia (Ramaekers et al.. 1987). CK13 expressed in the most proximal cell layers of cell islands and cell columns is usually found in stratified internal epithelia, where it is a suprabasal marker indicative ofadvanced differentiation (Kuruc et al., 1989; Sun et al., 1985). Only in the transitional epithelium does CKI3 show an expression pattern comparable to extravillous trophoblasts of cell islands and cell columns. since in the urothelium this cytokeratin is present in cells of the basal and intermediate layers (Moll et al., 1988). From these data it becomes evident that in cell islands and cell

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Figure 5. Two frozen, parallel sections of a full-term placenta stained by immunoflourescencetechniques with (A) antibody E 3 (anti-CW) and with (B) anti-fibrin antibody. Patchy CK17 positivity (A) is found in a larger villus next to fibrin deposit (E). f. fibrin deposit. Bar = 100 pm.

columns the CK pattern is not similar to the pattern in the typical stratifiedlmultilayered epithelia of human tissues. This can be due to the peculiar expression of basement membrane molecules as discussed above, but also to at least one additional reason. There is evidence that substances such as vitamin A, calcium, or growth factors such as EGF may influence CK expression in epithelial or mesothelial tissues (Yuspa et al., 1989; Fuchs et al., 1987; Kim et al.. 1987; Fuchs and Green, 1981). Concerning growth factors, it has been pointed out that in cytotrophoblastic cell islands and cell columns EGF receptors (EGF-R) are located in the cells proximal to the villous stroma, whereas the distal cells are EGF-R-negative (Muhlhauser et al., 1993; Bulmer et al., 1989). It is possible that EGF as well as other factors may act differently on the extravillous cytotrophoblast of cell islands and cell columns through the presence or absence of their specific receptors and that such factors may thus influence the expression of different cvtokeratins. A fundamental characteristic of the extravillous cytotrophoblast in the cell columns is its capacity to invade the decidua and to migrate deeply into the uterine wall. In their invasive behavior, these cells share several aspects with malignant cells (Ohlsson, 1989). As considered above, CK8 and CK18 are characteristic keratins of sim-

ple epithelia and are very weakly expressed or even absent in stratified epithelia under normal conditions (Markey et al.. 1991; Bosch et al., 1988). When the latter epithelia undergo malignant transformation. CK8 and CKl8 become strongly expressed (Markey et al., 1991; Schaafsma et al., 1990; Debus et al., 1984). This alteration in the expression of CK8 and CK18 has been considered to be related to some functional modifications of the malignant phenotype, such as its invasive capacity (Markey et al., 1991). This latter concept is emphasized by the observation that the invasive ability of transfected mouse L-cells increases when these cells express CK8 and CK18 (Chu et al.. 1990) and by recent studies indicating an increased expression of CK8 and CK18 in the invasion front of mucosal squamous-cell carcinoma (Schaafsma et al., 1993). It is possible that the presence of CK8 and CK18 in the cytotrophoblastic cell columns plays a role, together with other specific factors, in the invasiveness of the extravillous trophoblast into the uterine wall. This possibility is also supported by the fact that CK8 and CK18 are the earliest keratins expressed in the pre-implantation embryo (Jackson et al., 1980) but are subsequently lost from most differentiating tissues (Markey et al., 1991). In human placenta they are present in undifferentiated as well as differentiated trophoblas-

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Figure 6. Placentalvilli and cytotrophoblastic cells islands from first-trimester placenta. vs, villous stroma. Immunofluorescence staining patterns using the antibodies (A) Ks 7.18 (anti-CK7), (6) CY 90(anti-CK19) and (C) Ks 13.1 (anti-CK13). In A and B all extravillous cytotrophoblasticcells of the cell islands are positive. (C) Note the positive reaction product of the extravillous cytotrophoblastic cells (arrowheads) located proximal to the villous stroma and of the basal plasma membrane of the syncytium (arrow). Distally located extravillous cytotrophoblastic Cells (EC) are very weakly stained or negative. Bars = 20 um.

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Figure 7. Serial sections through the basal plate of a &week placenta embedded in paraffin. Streptavidin-biotin technique. A placental villus is anchored to the basal plate by a cell column. bp. basal plate. (A) lmmunostaining with MAC CY90 (antiCK19). All extravillous cytotrophoblastcells are positive. (B) Immunostainingwith MAb Ks 7.18 (antiCK7). Extravillouscytotrophoblast is positive in the cell column and in the most superficial layers of the basal plate. Immunoreactivity is ceasing in cells with increasingdistance from the cell column. Cells in profound areas of the basal plate are negative. (C) lmmunostainingwith MAb Ks 13.1 (antiCK13). The most proximally located extravillous trophoblast of the cell column is positive. Other extravillous trophoblastic cells located in the decidua are negative. Bar = 20 rm.

Figure 8. Frozen sections of the human amnion of (A) first-trimester placenta and (B) at term immunostained with antibody Ks 13.1 (antiGK13). (A) The amnion, folded on itself due to preparationtechniques, is completely negative. (B) At term the amnion epithelium shows an intense immunostaining. Arrow, amnion epithelium. Ear = 20 pm.

the different trophoblastic components throughout pregnancy. In addition, they provide a foundation for studying different pathological conditions, such as the placental site trophoblastic tumor, choriocarcinoma, or invasive mole (Mazur and Kurman, 1987). in which alterations in the differentiative or proliferative processes of trophoblast populations play a fundamental role.

Acknowledgments
subsets, i.e., these two CKs are not related to differentiation processes. This has also been suggested in an elegant in vitro study (Yamamoto et al., 1990)showing that suppression of CK8 mRNA in cytotrophoblastic cells does not influence their fusion and differentiation. Therefore, it is possible that CK8 and CK18 are involved in the invasive behavior of the extravillous trophoblast, having other functions in the villous trophoblast. In conclusion, our data represent a comprehensive analysis of the striking changes in the distribution patterns of cytokeratins in
We are gratehito Prof R. MoUfor usefdsuggestions. We than&Mr R. Ribani for exellent technicalassistanceand Mr M. Rucifor the photographic work.

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H. Kopinski M (1986): The influence of protease digestion and

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