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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

January 2003
Volume VI, Number 1
ISSN: 1537-6583
Pages: 214-234

Fibronectin Isoforms In The Extracellular Matrices Of Human Term Placenta*

Ayşe Y. Demir^1,2

(1) Department of Biochemistry, Medical Faculty, Akdeniz University,Antalya, Turkey; (2) Research Institute Growth and Development (GROW), Academic Hospital and Maastricht University, Maastricht, The Netherlands

Short title: Fibronectin isoforms in human term placenta

Correspondence address: Ayşe Yasemin Demir, MD, PhD, La Traviatadreef 30, 3561 KS Utrecht, The Netherlands, Telephone: 0031-30-2656770, e-mail: demirweusten@yahoo.com

*This paper is partly adapted from Dr. Demir‘s PhD thesis entitled as "Extrazellulaere Matrices in der reifen menschlichen Placenta: Verteilung von Fibronektin-isoformen und Matrixmetalloproteinasen", which was submitted at RWTH, Aachen, Germany

Acknowledgements: A couple of people are greatly acknowledged; Prof. Dr.P. Kaufmann, Dr. B. Hupertz for their excellent supervising during the preparation of the PhD thesis, Uta Zahn for her excellent technical support and Gaby Bock for her expertise photographical assistance. This study was performed at Department of Anatomy, University Hospital of Aachen, Germany. Dr. Ayşe Y. Demir was sponsered by Turkish Scientific and Technical Research Foundation (TÜBİTAK) as a NATO-A1 Fellow.

In this study we aimed to analyze the distribution of fibronectin isoforms in the human term placenta and to evaluate the differences between the matrices produced by fetomaternal tissues.

Tissue samples were collected from different sites of human term placenta such as amnion, basal plate, chorionic plate, decidua, placental fibrinoid, umbilical cord and villi. By using immunohistochemistry, SDS-PAGE and Western blotting methods the expression of fibronectin isoforms (general, cellular and oncofetal) was determined and compared.

In nearly all tissues reactivities for fibronectin isoforms were determined. Strongest reactivities for cellular and oncofetal fibronectins were found in the areas such as amnion, basal plate, chorionic plate and chorion laeve.

Our results show that fibronectin molecule is one of the building elements of human term placenta. The cellular and oncofetal isoforms are located at the placental sites composed of mainly invasive trophoblast cells, indicating the importance of these isoforms in trophoblast differentiation, invasion and adhesion. Furthermore the presence of these isoforms at sites where no invasive trophoblast cell exists imply their involvement in high tissue turnover due to rapid growth of fetus and its membranes.

Fibronectins are adhesive mosaic glycoproteins comprised of three general types of homologous iterative modules. These modular units are used as basic building blocks to form domains that develop distinct functions such as establishment and maintenance of normal cell morphology, cell migration, homeostasis and thrombosis, wound healing and oncogenic transformation (Kornblihtt et al., 1985).

Although fibronectin is encoded by only one gene, this protein exists in a number of variant isoforms due to alternative splicing and/or posttranslational modifications. These comprise modifications such as glycosylations and splicing at three general regions of the precursor mRNA; IIICS, ED-A and ED-B (Johansson et al., 1997). The plasma fibronectin isoform lacks the alternatively spliced domains, whereas the cellular fibronectin isoform contains the ED-A domain in their protein (Zardi et al., 1987).

In transformed cells and malignancies the alternative splicing of the primary transcript of fibronectin is deregulated (Borsi et al., 1992). The fibronectin isoforms containing the domains III-A (ED-A), III-B (ED-B) and III-V (IIICS) are expressed to a higher degree in transformed human cells and in tumor tissues than in their normal counterparts (Castellani et al., 1986, Carnemolla et al., 1992) . Especially, fibronectin containing the III-B sequence is detectable almost exclusively in healing wounds, in fetal tissues and in tumor tissues (Ffrench-Constant et al., 1989, Laitinen et al., 1991).

In the human placenta, fibronectin molecule is detectable in various sites such as amnion, basal plate, chorionic plate, decidua, placental fibrinoid, umbilical cord and villi. Although the presence of all of fibronectin isoforms are not studied in the extracellular matrices (ECMs) of all these sites, the special matrix of invasive extravillous trophoblast (EVT) cells, matrix-type fibrinoid, is particularly evaluated in detail. Huppertz et al. (1996) showed that the patches of fine fibrillar networks of matrix-type fibrinoid were immunocytochemically reactive with general (IST-4, IST-6), cellular (IST-9) and oncofetal isoforms (BC-1, FDC-6) of fibronectin. These molecules especially oncofetal isoforms are addressed to play roles in the differentiation of trophoblast cells from a proliferative subtype to an invasive subtype.

The aim of this study was to present the differences between matrix-type fibrinoid and the other matrices from fetal or maternal origin. For this purpose, different extracellular matrices were extracted from various regions of human term placenta. Following extractions, the distribution of fibronectin isoforms within these extracellular matrices was evaluated and compared using immunohistochemical and biochemical techniques.

Immunohistochemistry
Tissue samples from 7 term human placentas were used. Placental tissue in the forms of cubes with a maximal edge length of 20x20x5 mm³ were fixed in phosphate-buffered neutral 4% formaldehyde solution for a maximum of 24 hours at 4° C. The specimens were dehydrated in a graded series of ethanol and embedded in paraffin (melting point 52° C; Merck, Darmstadt, Germany) using xylene as intermedium, not exceeding a temperature of 58° C. Serial sections (3-5 µm) were cut and mounted on glass slides. The sections were then deparaffinized using xylene and a graded series of ethanol (10 minutes each step).

Immunohistochemical staining was performed according to a standardized sequence as described before (Frank et al., 1994). Briefly, endogenous peroxidases and nonspecific binding were blocked with 3% hydrogen peroxide in methanol and with swine serum (1:20) in 0.05 M Tris-HCl, pH 7.6, 15 mM sodium azide, 6% BSA, respectively. After incubating with primary antibodies (Table 1) in 0.05 M Tris-HCl, pH 7.6, 15 mM sodium azide, 12.5% BSA, a biotinylated link antibody (Dako, E453, swine antibody, 1:25, Hamburg, Germany) was applied for 30 minutes. Subsequently, the binding of streptavidin-horseraddish-peroxidase (Dako, P397, 1:400) to the antibody-antigen complexes was detected with AEC chromogen.

Tissue preparation
Human term placentas (n=7) were collected from clinically normal pregnancies terminated by either vaginal deliveries or cesarean sections. After their immediate transfer on ice, samples (5-10 g) from different regions (amnion, basal plate, chorionic plate, decidua, chorion laeve, Nitabuch’s fibrinoid, umbilical cord, villi) were cut out as precisely as possible, put into 5% sucrose in PBS, pH 7.4, frozen in liquid nitrogen, and stored at -35° C until further use.

Extraction procedure and protein determination
The extraction of matrix proteins was performed on ice in the presence of protease inhibitors (Protease inhibitor cocktail, Boehringer, Ingelheim, Germany) as it can be seen in Table 2.

Before electrophoresis the protein concentration of each aliquot was determined using an Elisa reader (using Microwin or Easywin software program), at either 620 nm with Bradford (Biorad, Munich, Germany) or 700 nm with DC-Lowry (Biorad) protein estimation assay.

SDS-polyacrylamide gel electrophoresis and western blotting
SDS-polyacrylamide gel electrophoresis was carried out under reducing conditions according to Laemmli (1970). The 7% separating gel was overlayered with a stacking gel containing 3% polyacrylamide. Samples with the same protein concentration (15-20 µg/per lane), were incubated for 30 minutes at room temperature in sample buffer made up of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% sucrose, 0.005% bromphenol blue and 5% 2-mercaptoethanol. Before loading the samples, they were centrifuged for 10 minutes at 10000 xg. The proteins were separated at 25 mA / plate in 25 mM Tris-HCl, pH 6.8, 192 mM Glycine, 0.1% SDS for approximately 1.5 hour until the dye front reached the end of the gel. After electrophoresis the gels were either stained with Coommassie blue R-250 or subjected to semi-dry transfer on PVDF membranes (Westran, 0.2 µm, Schleicher & Schuell, Dassel, Germany) at 200 mA for 20 minutes according to Towbin (1979). Following the transfer of proteins, blotted membrane were blocked with 5% nonfat dry milk in 0.05% Tween 20 / PBS, pH 7.4 for 2 hours and incubated with the primary antibodies (Table 1) overnight and developed using gold labeled goat anti-mouse IgG or IgM for 2 hours and a silver enhancement kit for 40 minutes (Amersham Life Sciences, Braunschweig, Germany). The immunoreactivities were evaluated by gel print 2000I (Biophotonics corporation, MWG-Biotech, Germany) and scananalytics (Billerica, Germany) (for details see Demir Weusten, 1999).

IST-4 (III-5 domain of all fibronectin isoforms)
Amniotic basal lamina was very faintly immunoreactive with this antibody, whereas the amniotic epithelium and mesoderm showed no immunoreactivities. Chorionic mesoderm was also faintly immunoreactive. The EVT cells in the chorionic plate, in the appearances like cell islands between the villi and cell columns and in the basal plate showed quite remarkable, patchy like distribution in their matrices (Figures 1A-C). In the cell columns immunoreactivity increased from proximal to distal parts. ECM of decidual cells in the basal plate was also immunoreactive. The stroma of large stem villi showed immunopositivity, whilst trophoblastic cover remained immunonegative. The walls of big artery walls in these stem villi also showed no immunoreactivity. Other types of villi having smaller caliber showed immunopositivity neither in the stroma nor in the trophoblastic cover (data is not shown).

IST-6 (III-7/8 domain of fibronectin lacking III-B domain)
In amnion, amniotic epithelium, basal lamina and mesoderm were not immunoreactive with this antibody. Chorionic mesoderm was very faintly immunoreactive. The EVT cells in the chorionic plate, in the appearances like cell islands between the villi and in the cell columns of the basal plate showed similar appearances as obtained by IST-4 (Figures 2A-C). The matrix of decidual cells in the basal plate were also immunoreactive (Figure 2D). In stem villi, the stroma was immunopositive, the walls of the big arteries gave very faint reaction and the trophoblastic cover was immunonegative. Other types of villi showed immunopositivity neither in the stroma nor in the trophoblastic cover.

3E1 (heparin binding domain of all fibronectin isoforms)
Amniotic epithelium showed no immunoreactivities, but basal lamina underlying the epithelium and amniotic mesoderm was immunoreactive with this antibody. Chorionic mesoderm was faintly immunoreactive and EVT cells in the chorionic plate were rarely immunopositive. The stroma of stem villi and anchoring villi were immunoreactive, whereas the stroma of the other types of villi was faintly immunoreactive. The walls of the big arteries were immunopositive. In the basal plate there were spot like irregular immunoreactivities. The trophoblast cells in cell islands were not immunoreactive. In the basal plate decidua cells were very almost immunonegative.

3E3 (cell binding domain of cellular fibronectins)
This antibody was immunoreactivity with amniotic basal lamina and mesoderm, but was not reactive with amniotic epithelium. Chronic mesoderm was immunopositive, but trophoblast cells did not show reactivities. Stroma of villi was immunoreactive, whilst trophoblastic cover of villi did not show this appearance. In the basal plate stroma of anchoring villi and trophoblast cells were very weakly reactive. Decidual ECMs was also very weak positive.

IST-9 (III-A domain of cellular fibronectins)
IST-9 showed reactivities very similar to that of IST-4 and IST-6. Amnion in all parts showed no immunoreactivities, whereas the border line of chorionic mesoderm was very weakly reactive and the patchy like distribution of the reactivity in the ECM of EVT cells was remarkable. The stroma of stem villi were very weakly immunoreactive, but their trophoblastic cover was not reactive. Other types of villi showed immunopositivity neither in the stroma nor in the trophoblastic cover, but the appearances like cell islands between the villi were immunoreactive. Cell columns in the basal plate containing EVT cells showed also patchy like distribution in their ECMs. The ECMs of decidual cells and EFVT cells in the basal plate were also immunoreactive.

FDC-6 (O-glycosylated III-V domain)
FDC-6 was reactive with amniotic basal lamina, whereas was not reactive with amniotic mesoderm and amniotic epithelium (Figure 3A). Chorionic mesoderm showed very faint reactivity, while the EVT cells of the chorionic plate had the typical appearance of the patch work distribution in their ECMs (Figure 3B). The stroma of the stem villi and of the other types of villi and their trophoblastic cover showed no immunoreactivities. The cell islands between chorionic villi tree, EVT cells in basal plate, and also the decidual cells showed reactivities in their ECMs (Figure 3C).

The results from immunohistochemical reactions are summarized in Table 3 semiquantitativly and the corresponding reactions are given in representative figures.

Biochemistry
Fibronectins in extracellular matrices of human term placenta were solubilized by the application of extraction buffer (Table 2) and the supernatants that were obtained at this last step were applied on a SDS-PAGE (Figure 4) followed by the quantification of fibronectin isoforms on Western blots. The signal of each fibronectin isoforms were analyzed and compared by the integration of detected optical densities (int OD) (Table 4).

IST-4 (III-5 domain of all fibronectin isoforms)
The immunoreactivities of this antibody gave the strongest signal with the chorion laeve extract, which was then pursued by the preparations from amnion and chorionic plate. The extracts from basal plate, villi, umbilical cord and decidua were reactive with IST-4 less intense, but had very close densities to each other. The weakest signal was detected in the preparations of and Nitabuch’s fibrinoid (Figures 5A-G and 6A-G).

IST-6 (III-7/8 domain of fibronectin lacking III-B domain)
The most intense immunoreactivities with this antibody were detected in the preparations of amnion and chorion laeve. The density of chorionic plate extract was very close to the densities detected in the extracts of amnion and chorion laeve. The preparation from basal plate extract had an intenser immunoreactivity than the preparations from decidua, umbilical cord and Nitabuch’s fibrinoid. While these last three extracts had very close reactivities, the preparation from villi showed the weakest immunoreactivity (Figures 5A-G and 6A-G).

3E1 (heparin binding domain of all fibronectin isoform)
The immunoreactions of this antibody showed similar intensities with different extracts. Among them the extracts from chorion laeve and amnion were the leading dense signals. The extracts from chorionic plate and Nitabuch’s fibrinoid had the same level of detected density. Umbilical cord followed the last two with very close intensity. The rest of the sequel was formed by the preparations from basal plate, decidua and villi (Figures5A-G and 6A-G).

In addition, another immunoreactive band, around 41-43kD region was detected.

4B2 (gelatin binding domain of all fibronectin isoforms)
This antibody was intensely immunoreactive with the preparation from chorion laeve, which was then followed by amnion, chorionic plate, villi, umbilical cord, basal plate and Nitabuch’s fibrinoid (Figures 5A-G and 6A-G).

Cellular fibronectins

3E3 (cell binding domain of cellular fibronectins)
This antibody like the other fibronectin antibodies was strongly immunoreactive with the extract from chorion laeve, which was followed, by the extracts of amnion and chorionic plate. Intensity signals from the preparations of decidua and Nitabuch’s fibrinoid were close to each other and were denser than basal plate and villi (Figures 5A-G and 6A-G).

IST-9 (III-A domain of cellular fibronectins)
IST-9 showed the strongest immunoreactivity with the extract of chorion laeve, which was prosecuted by the signals from the preparations from chorionic plate and Nitabuch’s fibrinoid. The densities of the last two extracts were near to each other and were higher than the signals of amnion and umbilical cord, which were also at the same intensity level. The densities detected from the extracts of villi, decidua and basal plate completed the sequel with their lower intensities (Figures 5A-G and 6A-G).

FDC-6 (O-glycosylated III-V domain)
FDC-6 was drastically immunoreactive with the extract from chorion laeve. The preparations from amnion and chorionic plate had close intensities and pursued the signal of chorion laeve. The extracts from decidua, Nitabuch’s fibrinoid, basal plate, villi and umbilical cord followed the first three signals from high to low intensities (Figures 5A-G and 6A-G).

In this study we have shown the distribution of fibronectin and its isoforms in different human placental sites such as amnion, basal membrane, chorionic plate, decidua, chorion leave, Nitabuch’s fibrinoid, umbilical cord and villi. Our results demonstrate the features of fetoplacental and decidual tissue components influenced the amount and type of fibronectin detected.

At the first comparative analysis of the results obtained by using immunohistochemistry and Western blotting gives the impression as if some of the results are not complementary. The following pionts may be the underlying factors for the differences between the immunoreactivities obtained in both of the methods.

(a) occupied epitopes: During the matrix organization and assimilation, fibronectin molecules interact with other matrix proteins and with cells (Aplin and Hughes, 1982). As a result, some of the epitopes can be occupied making it impossible for several anti-fibronectin antibodies to bind during immunohistochemistry. After application of different denaturants, possibly these antigenic determinants are evacuated. The very weak reactivities for 3E1 (heparin binding site) and 3E3 (cell binding site) in immunohistochemistry can be due to the occupied epitopes.

(b) denatured epitopes: The extraction steps might lead to denaturation of epitopes on the fibronectin molecule in some cases.

(c) altered three dimensional structure: Insertion of an extra type III module such as III-A into an array of repeated type III modules rotate fibronectin up to 180° at the position of the insertion (Manabe et al., 1997). This leads to an alteration in the conformation of fibronectin molecule.

The weak reactivities obtained with IST-4, IST-6 and IST-9 could be due to denature epitopes and/or altered three dimensional structure.

(d) acquisition of the extraction method: Different placental sites have different ECM compositions and organizations, due to the different cellular compositions and functions. As a result of this, the ECM that could be extracted with the methods used can vary. Although in immunohistochemistry one antibody shows comparable binding patterns and reactivities in two different matrices, the binding patterns can be completely different after extraction steps.

The results of immunohistochemistry experiments have revealed that amniotic basal lamina is mainly formed by fibronectins having III-5, heparin binding, cell binding and O-glycosylated III-V domains. Amniotic mesoderm is formed by fibronectins having heparin and cellular binding domains. Western blots demonstrated that amnion is also composed of fibronectins having III-7/8 and III-A domains. In previous studies, the presence of fibronectin in amnion was reported (Aplin and Campbell, 1985), while human amnion epithelial cells were shown to secrete cellular (III-A domain containing) and oncofetal (III-B containing) fibronectin isoforms that are assembled in the ECM (Vartio et al., 1989, Linnala et al., 1993). These results suggest that fibronectins in the amniotic basal lamina, particularly oncofetal isoform, is a part of healing process in this tissue due to the rapidly growing fetus causing partly shears in the amnion.

In basal plate and chorionic plate three isoforms of fibronectin; familiar, cellular and oncofetal, were detected by using both immunohistochemistry and Western blotting. Especially, in distal parts of the cell columns and in the trophoblastic layer of the chorionic mesenchyme, these isoforms were distributed in a spot like manner between the EVT cells in agreement with previous reports (Frank et al, 1985, Vicovac et al., 1995, Huppertz et al., 1996).

Trophoblastic layer of chorionic plate can also represent chorion laeve, although immunohistochemistry results were not available for this site. In Western blots chorion laeve extracts gave the highest signals with general, cellular and oncofetal fibronectins compared to other placental sites. The high concentrated numbers of EVT cells and their matrices in this region might lead to this finding. Especially, the presence of oncofetal fibronectins in the regions where the EVT cells are in great numbers, suggest an important role at the chorio-decidual interface together with the other fibronectin isoforms (Feinberg and Kliman, 1993).

Decidual tissue was taken from the basal plate for immunohistochemistry, while decidual tissue for Western blots was derived from the fetal membranes. In both techniques the results were parallel to each other revealing the presence of fibronectin isoforms; general, cellular and oncofetal, in decidual tissue. In a previous study the presence of fibronectin in the pericellular basement membrane (Wewer et al., 1985), in the pericellular matrix (Korhonen and Virtanen, 1997) and in decidual cell cytoplasm in fibrillar and punctuate patterns (Kisalus et al., 1987) have been shown. Interestingly, oncofetal fibronectins having III-B or O-glycosylated III-V domain have been detected only in decidua that has been invaded by the trophoblast (Feinberg et al., 1991, Korhonen and Virtanen, 1997).

The presence of fibronectins at fetomaternal contact regions suggest that trophoblast adhesion to maternal tissues is enhanced, trophoblast migration is facilitated and trophoblast survival is improved (Pijnenborg et al., 2000). The presence of oncofetal fibronectins in particular is a connected to the differentiation of proliferative trophoblast cells into an invasive type. Furthermore, local pH is an important regulator of this differentiation process (Gaus et al., 2002).

A variety of polypeptides having molecular weights over 105kD have been reported in Nitabuch’s fibrinoid, besides b chain lacking fibrinopeptide, (Sutcliffe et al., 1982). Among these polypeptides, basement membrane proteins (collagen type IV and laminin) have been shown in this region (Fernandez et al., 1992). In addition to these reports, the results of the present study revealing the presence of different general and cellular fibronectins indicates their important role in the control of placental growth and trophoblast invasion.

The synthesis of fibronectin by the venous and arterial endothelia of the human umbilical cord has been previously reported (Levene et al., 1988). The results of the current study presenting general and cellular fibronectin isoforms in umbilical cord extracts imply that these molecules are heavily involved in the stabilization of maternofetal dialogue.

According to previous studies in term placenta, the stroma of chorionic villi (Isemura et al., 1985, Amenta et al., 1986), the stroma of big stem villi (Demir et al., 1997) and the surrounding of fetal vessels (Virtanen et al., 1988) are immunoreactive for fibronectin, while the trophoblastic basement membrane is not (Yamaguchi et al., 1985). Furthermore, oncofetal fibronectin that has III-B domain is present in the villous stroma (Nanaev et al., 1993, Nanaev et al., 1997). In addition to these reports current study shows that general and cellular fibronectin isoforms are the predominant subtypes in these placental sites and oncofetal fibronectin isoform that has O-glycosylated domain is also present in the villous stroma. These results suggest that general and cellular fibronectins are involved in the placental villi architecture, whereas oncofetal fibronectins are likely located at the regions where villi growth and development still continue.

In previous reports fibronectin isoforms have been implicated in physiological and pathological stituations (Moore, 1999, Kramer et al., 2001). Oncofetal fibronectin, in particular, is a very good candidate to become a clinical indicator or predictor of true labor, preterm labor, or some complications of pregnancy (Dunn and Feinberg, 1996). Alterations in levels of oncofetal fibronectin occur in preterm labor, premature ruptured membranes, postterm pregnancy, and pregnancy-induced hypertension (Feinberg and Kliman, 1992) (Guller et al., 1995) (Moore, 1999) (Lockwood et al., 1991).

In conclusion different fibronectin isoforms are considered as very important proteins determining the success of implantation and survival of placenta. Knowledge about the distribution of fibronectin isoforms, therefore, has implications for the interpretations of clinical pregnancy associated situations.

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The antibodies (monoclonal) that were used in this study. Dilutions used for *immunohistochemistry and **Western blots. FN: fibronectin

Extraction procedure. Lysis buffer: 100 mM Tris-maleic acid, pH 6.8, 5 mM EDTA, 5 mM DTT. Washing buffer: 2% Triton X-100 added in lysis buffer. Extraction buffer: 50 mM Tris-maleic acid, pH 6.0, 8 M guanidine, 5 mM EDTA, 5 mM DTT, 1 mM PMSF, 10 M ethylmaleimide.

Semiquantitative evaluation of the immunoreactivities in different placental regions. GFn, CFn, Ofn: General, cellular and oncofetal fibronectins, respectively. +/-: faint reaction, +: positive reaction, ++: remarkable reaction, ~: unregular reactivity, *: not determined. A: amnion (e: epithelium, bm: basal membrane, m: mesenchyme); Bp: basal plate (D: decidua, ET: extravillous trophoblast cells in basal plate); Cp: chorionic plate (m: mesenchyme, ET: extravillous trophoblast cells in chorionic plate); Cl: extravillous trophoblast cells in chorion laeve; Ci:extravillous trophoblast cells cell islands; CC: extravillous trophoblast cells in cell columns; V: different types of villi (Av: anchoring villi, Sc: small calibrated villi, Bc: big calibrated villi, S: stroma, Ts: Trophoblastic shell, Ba: walls of big arteries).

The comparison of immunoreactivities in different extractions. The reactivities obtained by each antibody are given in the horizontal direction and can be compared with each other. The values in the vertical direction can not be compared, because for each antibody a separate SDS-PAGE and Western blot is performed, thus the degree of the reaction differs.

These micrographs show the immunoreactivities with IST-4 in the ECMs of EVT cells in A: chorionic plate (CP), B: cell islands (CI) and C: basal plate (BP). AM: amniotic membrane. Original magnification is x 25.

The immunoreactivities with IST-6 in A :chorionic plate (CP), B: cell islands (CI), C: cell columns (CC) and D: decidual (D) part of the basal plate (BP) are shown. AM: amniotic membrane; AV: anchoring villi. Original magnification is x 25.

The immunoreactivities with FDC-6 in A: amniotic membrane (AM), B: chorionic plate (CP) and C: basal plate (BP). Amniotic membrane is immunonegative, except a weak immunopositivity in the basal lamina of amniotic epithelium. However, ECMs of EVT cells in CP and BP have strong positive signal for FDC-6 antibody. Chorionic mesenchyme (MZ) is immunonegative. Original magnification x25.

A representative SDS-PAGE with the extracts from different sites in human term placenta. This application design is also used for Western blots. From left to right; molecular weight markers, extractions from amnion (a), basal plate (b), chorionic plate (c), decidua (d), chorion laeve (e), Nitabuch’s fibrinoid (f), umbilical cord (g) and villi (h).

Representative Western blots from the extractions of different placental sites with antibodies against different fibronectin isoforms. The order of blots is similar to that in figure 4. Immunoreactivities of different levels are seen with IST-4 (A), IST-6 (B), 3E1 (C), 4B2 (D), 3E3 (E), IST-9 (F), and with FDC-6 (G).

The integrated values of optical densities (y-ordinate) are compared with the extracted tissues (x-ordinate). The strongest immunoreaction is considered as 100% and the other reactions were carried out as <100%. The order of x-ordinate is similar to that in figure 4. The immunoreactivities for fibronectin isoforms IST-4 (A), IST-6 (B), 3E1 (C1 and C2), 4B2 (D), 3E3 (E), IST-9 (F), and with FDC-6 (G) are shown.