051201.500 161201.200 132119.426 010408.500 200112.500 130114*142

Elastic fibres are an essential componentof human placental stem villous stroma and an integrated partof the perivascular contractile sheathR. Graf1, H. Neudeck1, R. Gossrau1, K. Vetter2

1 Department of Anatomy, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Königin-Luise-Strasse 15,D-14195 Berlin, Germany2 Department of Gynecology and Obstetrics, Krankenhaus Neukölln, Mariendorfer Weg 28, D-12051 Berlin, Germany

&misc:Received: 2 May 1995 / Accepted: 7 August 1995

&p.1:Abstract. The stroma of human placental stem villi isbelieved to consist only of reticular and collagen fibres.In the present study we were able to show for the firsttime by light (orcein staining) and electron microscopylarge amounts of elastic fibres in the stem villous stro-ma. Electron microscopically, homogeneous elastin wasfound alone or in association with microfibrils. In addi-tion, microfibrils were observed forming long bands.These three structures, generally known to form elasticconnective tissue, were seen in close connection withplacental extravascular smooth muscle cells, which be-long to the perivascular contractile sheath (PVCS) ofstem villi. Elastin was associated with these smoothmuscle cells and connected to collagen fibres via micro-fibrils. Collagen fibres were additionally interconnectedby spike-like structures. Extravascular smooth musclecells revealed numerous adhesion plaques which occu-pied conspicuously long cytoplasmic faces of the plasmamembrane. In cryostat sections, immunoreactivity oftalin, an attachment protein of adhesion plaques linkingintracellular α-actin filaments with extracellular fibro-nectin, was detected in extravascular and vascular (me-dia) smooth muscle cells. The arrangement of placentalextravascular smooth muscle cells, elastic and collagenfibres suggests a functional myofibroelastic unit withinthe PVCS, which surrounds the large foetal blood ves-sels possibly contributing to elasticity and supportingtensile and/or contracting forces within the stem villi.

&kwd:Key words: Elastic fibres – Placental stem villi – Extra-vascular smooth muscle cells – Adhesion plaques – Talinimmunoreactivity – Human

Introduction

It has been established that the stroma of the human pla-centa and especially that of stem villi is composed solelyof reticular and collagen fibres (for reviews, see Boydand Hamilton 1970; Schiebler and Kaufmann 1981;Benirschke and Kaufmann 1990). Interestingly, microfi-brils, generally being defined as part of the elastic con-nective tissue (see below), were isolated from the pla-centa and interacted with platelets in a similar way asaortic microfibrils (Legrand et al. 1986). Microfibrilswere also localised ultrastructurally in the trophoblasticbasement membrane of terminal villi (Arbeille et al.1991). In the present study we demonstrate for the firsttime by light and electron microscopy elastic connectivetissue to be a substantial component of the stroma in hu-man placental trunci and rami chorii (according to theclassification of Kaufmann et al. 1979).

Materials and methods

Tissue preparation

Human placentae of uncomplicated pregnancies were collected af-ter spontaneous delivery or Caesarean sections for reasons of mal-position between 39 and 41 weeks of gestation.

Transmission electron microscopy. &p.2:Immediately after delivery ofthe placenta, allantois blood vessels were followed from the chori-onic plate into the depth of the placenta by forceps preparation.Trunci and rami chorii were dissected by coarse removal of sur-rounding villous tissue and immersed in Karnovsky’s solution for24 h at 4°C. Subsequently, the perivascular contractile sheath(PVCS) was dissected as described previously (Graf et al. 1995)and tissue samples with an average edge length of 2 mm werefixed for another 24 h in the same solution at 4°C. After postfixa-tion in 1% OsO4 in phosphate buffer, pH 7.2, for 24 h at 4°C, thetissue samples were processed routinely in a graded series of etha-nol followed by propylene oxide as intermedium and embeddingin Epon-Araldite. For orientation, semithin sections were stainedwith toluidine blue. Ultrathin sections were double stained with4% uranyl acetate (20 min) and 10% lead citrate (10 min) and ex-amined in a Zeiss 109 electron microscope.

This investigation was supported by grants from the DeutscheForschungsgemeinschaft (SFB 174)

Correspondence to:R. Graf&/fn-block:

Cell Tissue Res (1996) 283:133–141

© Springer-Verlag 1996

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Light microscopy. &p.2:Tissue samples 3–5 mm thick and 1–2 cm longwere collected 2–3 cm off the umbilical cord. For the preparationof cryostat sections, the samples were mounted on specimen hold-ers, wrapped in plastic foil, frozen in liquid nitrogen and stored at–40°C.

Staining procedures

Orcein staining. &p.2:5 to 10-µm-thick cryostat sections were cut at–25°C with a Reichert-Jung cryostat (model 2800 N, Heidelberg,Germany) and subsequently air-dried. The sections were stainedconventionally with freshly prepared acid orcein (Merck, Darm-stadt, Germany, Art.-No. 7091, Charge-No. 1369922) according toTaenzer-Unna (Unna 1891) as described by Romeis (1989): 1 gorcein was dissolved in 100 ml 70% ethanol and 1 ml 25% HClwas added. The solution is ready for use immediately after prepa-ration. Sections were stained for 1 h, 24 h and 48 h at room tem-perature and 56°C. After staining, the sections were rinsed in dis-tilled water and 96% ethanol and differentiated in 100% ethanol.Sections were then mounted in Eukitt (Kindler, Freiburg, Germa-ny).

Immunocytochemistry. &p.2:5-µm-thick cryostat sections were mountedon 0.01% poly-L-lysine (Sigma, Deisenhofen, Germany) -coatedglass slides and stored at –40°C. Before use, they were air-driedfor 24 h at room temperature, pretreated with acetone (5 min,–25°C), air-dried again and rinsed in phosphate-buffered saline(PBS), pH 7.6. The sections were used for immunostaining withmonoclonal antibodies against talin (1:200, clone TA 205, Novoc-astra Laboratories, Loxo, Dossenheim, Germany), fibronectin(1:800, clone FN15, Sigma) and α-actin (1:60, clone asm-1,Boehringer, Mannheim, Germany). Visualisation of binding siteswas performed with the ABC-Method (Vectastain ABC Peroxi-dase DAB-Kit, mouse IgG, Serva, Heidelberg, Germany) accord-ing to the instructions of the manufacturer, or using TRITC-la-beled goat anti-mouse IgG (Nordic, Tilburg, Netherlands). All in-cubation steps were performed in a humid chamber at room tem-perature. Incubations with specific antibodies were performed for30 min, with TRITC-labeled IgG for 45 min. Control reactionswere carried out using PBS instead of specific antibodies.

Source of chemicals. &p.2:If not otherwise stated, the chemicals werepurchased from Merck or Sigma.

Results

Transmission electron microscopy

Semithin sections of PVCS preparations used for orien-tation and trimming of ultrathin sections revealed differ-ent features. Either the preparations contained mainlysmooth muscle cells closely arranged to each other inlongitudinal direction and little connective tissue, or theyshowed large amounts of loosely undulating connectivetissue fibre bundles with weakly toluidine blue-stainedcells. Connective tissue and smooth muscle cells ran inparallel and were arranged longitudinal to each other. Noblood vessels were detected in semithin sections.

Ultrathin sections of regions rich in connective tissuecontained smooth muscle cells, fibroblasts and myofi-broblasts. Elastic fibres were shown to be a major com-ponent of stem villus connective tissue. Cells and theirprocesses with distinct plasma membranes were seen be-tween homogeneous elastic fibres, which in turn ap-

peared to be connected to collagen fibres via microfila-ments (Figs. 1, 2). Collagen fibres were obviously inter-connected to each other by spike-like structures (Figs. 2,5). The elastic material formed thick or thin fibres withor without connected microfilaments (Fig. 3). Further-more, some bundles of microfilaments with scanty par-ticipation of elastin formed very long and often undulat-ing bands running parallel to smooth muscle cells, myo-fibroblasts, fibroblasts and/or collagen fibres (Fig. 4), of-ten arranged as collagen and elastic fibre layers (Fig. 5).Contractile cells ended with long cytoplasmic processeswithin collagen or elastic fibres.

In the cytoplasm of smooth muscle cells of all PVCSpreparations filamentous areas dominated. The cells con-tained numerous dense and adhesion plaques, the latterof which mostly occupied large cytoplasmic areas of theplasma membrane (Fig. 6). Caveolae were often seen inassociation with the plasma membrane. Smooth musclecells were surrounded by a basal lamina. In PVCS prep-arations with closely arranged smooth muscle cells,these cells formed indentations almost to the full lengthof the cells. At the invagination sites an intact basal lam-ina was seen. Cell invaginations were arranged regularlyor depicted a pine-tree-like morphology with markedlylong cell processes arranged over long distances at regu-lar sharp angles. Extravascular smooth muscle cells alsorevealed regularly formed cell processes which ended inelastic or collagen connective tissue. Smooth musclecells were also arranged closely side by side withoutshowing any contacts. In these cases their plasma mem-branes were straight or only slightly invaginated and theadhesion plaques, which again were mostly unpaired(Fig. 6), extended over long distances of the plasmamembrane. Often electron-dense and lucent cells ofidentical structure were located adjacent to each other.Thin elastic and a few collagen fibres as well as microfi-laments were found between closely arranged smoothmuscle cells.

Orcein staining

After orcein staining, cryostat sections revealed manybrown-violet stained thick, thin or very delicate elastic fi-bres. Results were best after staining for 24 h or 48 h atroom temperature but stained fibres could already be de-tected after 1 h. Heating of the staining solution did notimprove the results. Generally, stem villi revealed elasticfibres in the PVCS, in the rather thick tunica media of ar-teries and veins as well as in the adventitia. However, inseveral of the tunica media formations, elastic fibres weremissing. Blood vessel walls, their adventitiae and sup-porting connective tissue were also stained in the chori-onic plate. In terminal villi, elastic fibres were not seen.

In the PVCS, extremely long and straight thick, thinand very delicate fibres were seen running in a longitudi-nal direction to the foetal blood vessels (Fig. 7). Some ofthe fibres ramified into long or short branches. Otherelastic fibres appeared as small undulating, parallel bun-dles arranged longitudinal to the blood vessels (Figs. 9,10). Elastic fibres were seen to contact the trophoblastic

basement membrane (Fig. 9). Occasionally, stained elas-tic fibres appeared to contact unstained connective tissuefibres. Unstained fusiform cells were observed runningparallel to the elastic fibres. In several stem villi, elasticfibres were not only present inside the PVCS, but alsoformed bundles between foetal blood vessels or joined toa blood vessel at one side. Furthermore, bundles of elas-

tic fibres were found in sections of stem villi which werefree of foetal blood vessels. Here, the fibres ran longitu-dinal to these villi and ended in the subtrophoblasticzone, obviously in the trophoblastic basement mem-brane.

Elastic fibres of the PVCS were often associated withthe elastic fibres in the adventitia of blood vessels,

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Figs. 1–5. Transmission electronmicroscopy of elastic fibres in prep-arations of human placental perivas-cular contractile sheaths. Ramichorii&/fig.c:

Fig. 1. Smooth muscle cell process(SMC) between elastin (E) and col-lagen fibres. Spike-like structureson collagen fibres (arrow). Bar:0.1µm. ×120 000&/fig.c:

Fig. 2. Connection between elastin(thick arrow), microfibrils (arrow-heads)and collagen. Collagenfibres are connected via spike-likestructures (thin arrow). Bar:0.1µm. ×120 000. Inset: Spike-likestructures on collagen fibres(arrows). Bar: 0.05µm. ×150 000&/fig.c:

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which were very delicate, short and loosely arranged be-tween unstained connective tissue fibres. In blood ves-sels of the chorionic plate, trunci and rami chorii with athick layered tunica media, elastic fibres formed a dis-tinct elastica interna. Intensively stained fibres were alsopresent between the smooth muscle cell layers. In pairedvillous blood vessels with a thinner tunica media, oftenonly one of the villous vessels stained for elastic fibres.

Immunocytochemistry

The results obtained after α-actin-immunostaining havealready been described in detail by Graf et al. (1994). Inthe present study, incubation with this antibody wasperformed to compare with talin and fibronectin immu-noreactivity. Smooth muscle cells of the media of foetalblood vessels, extravascular smooth muscle-like cells of

Fig. 3. Elastin (E), microfibrils(arrow) and collagen fibres. Bar:0.1µm. ×120 000&/fig.c:

Fig. 4. Bundles of microfibrils(MF) poor in elastin (arrow). Bar:0.1µm. ×120 000&/fig.c:

the chorionic plate as well as numerous extravascularsmooth muscle cells belonging to the PVCS were α-ac-tin-immunoreactive. Cell processes of α-actin-immuno-stained extravascular smooth muscle cells occasionallyappeared in small and short undulating bundles. Theywere seen to contact the subtrophoblastic layer, obvi-ously the trophoblastic basement membrane (Fig. 11).Extravascular smooth muscle cells could additionally

form marked bundles between the foetal blood vesselsor, independent of the PVCS, adjacent to one of theblood vessels. However, often it was difficult to detect aborder between anti-α-actin-positive extravascular andmedia smooth muscle cells. Anti-α-actin reactivity wasalso localised in fusiform cells of stem villi which werefree of foetal blood vessels. The fusiform cells ran in alongitudinal direction within the villi and ended in the

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Fig. 5. Layers of elastic (E) andcollagen fibres. Note the spike-likestructures on collagen fibres(arrow). Bar: 0.2µm. ×72 000&/fig.c:

Fig. 6. Transmission electron mi-croscopy of smooth muscle cells inperivascular sheath preparations.Ramus chorii. Filament-rich areas,dense plaques (thin arrows), adhe-sion plaques (thick arrows). Bar:0.2µm. ×72 000&/fig.c:

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Figs. 7–12. Cryostat sections of human placental stem villi&/fig.c:

Fig. 7. Orcein staining, 48 h. Elastic fibres in a perivascular sheath(arrow); blood vessel (x), syncytiotrophoblast (arrowhead). Bar:20 µm. ×500&/fig.c:

Fig. 8. Talin immunoreactivity. Extravascular smooth muscle cells(arrows), smooth muscle of tunica media (M). Bar: 100µm. ×160&/fig.c:

Figs. 9, 10.Orcein staining, 48 h; same section photographed intwo different planes. Perivascular sheath with elastic fibres (ar-

rows). Elastic fibres with contact to the subtrophoblastic layer(Fig. 9, arrowheads). Blood vessels (x). Bar: 20µm. ×500&/fig.c:

Fig. 11. α-Actin immuno-fluorescence. Short undulating bundlesof extravascular smooth muscle cell processes (arrow), some ofthem contacting the subtrophoblastic layer (arrowhead). Bar:20 µm. ×500&/fig.c:

Fig. 12. Talin immunoreactivity. Perivascular sheath with anti-talin-positive extravascular (arrow) and vascular (media, M)smooth muscle cells. Immunoreactivity is also present in the syn-cytiotrophoblast (arrowhead). Bar: 100µm. ×160

subtrophoblastic zone, apparently in its basement mem-brane.

In extravascular and vascular (media) smooth musclecells, talin immunoreactivity showed the same pattern asimmunostaining with anti-α-actin, however, less clearly(Figs. 8, 12). In addition, portions of the basal syncytio-trophoblast were immunoreactive. Fibronectin immuno-staining was localised in the subendothelial and partly inthe subtrophoblastic basement membranes and in themedia of foetal blood vessels. Anti-fibronectin alsoshowed a positive staining pattern in the PVCS (data notshown), which resembled strongly that of α-actin immu-noreactivity.

Control sectionsincubated without specific antibod-ies showed no immunostaining.

Discussion

The present study demonstrates elastic fibres to be asubstantial component of the placental stem villous stro-ma and perivascular contractile sheath (PVCS). ThePVCS was shown to consist of extravascular smoothmuscle cells and a myofibroblastic subpopulation in as-sociation with connective tissue (Graf et al. 1992, 1994,1995). Alfa-actin-positive smooth muscle-like cells andmyofibroblasts have also been described by Beham et al.(1988), Demir et al. (1992) and Kohnen et al. (1993) inthe stem villous stroma. The close association of extra-vascular smooth muscle cells, elastic and collagen tissuefibres and their arrangement in layers within the PVCSgive further support to the assumption that stem villi re-quire a certain elasticity in addition to tensile strengthand/or contractility. Contractility of a sheath surroundingthe large foetal blood vessels in human placental stemvilli has already been suggested by Dubreuil and Rivière(1932) and Spanner (1936).

At present, very few and some contradictory informa-tion is available about the general presence of elastic fi-bres in the human placenta. Nikolov and Schiebler(1973) reported for foetal blood vessels of the term pla-centa, that elastic fibres were present only in umbilicalveins but absent in placental vessels. In contrast, Hof-man (1967) and Boyd and Hamilton (1970) describedelastic fibres in placental blood vessels. Hofman (1967)demonstrated the arrangement of elastic fibres in umbili-cal vessels and those of primary stem villi, while gener-ally no elastic fibres were present in vessels of second-ary stem villi. However, he pointed out that elastic fibresin primary stem villous arteries can also be missing.This is in agreement with our results. Baccarani-Contriet al. (1989) found amorphous elastic fibres in walls ofwide vessels of peripheral portions of placental villi at16 and 24 weeks of gestation. The absence of elastic fi-bres is mentioned by Kanayama et al. (1985) for thechorionic plate. The current opinion for the placental vil-lous stroma seems to be that elastic fibres are absent;this has also been described by Spee (1915). Connectivetissue is thought to consist only of reticular and collagenfibres (for reviews, see Boyd and Hamilton 1970;Schiebler and Kaufmann 1981; Benirschke and Kauf-

mann 1990). Microfibrils are generally defined as beingpart of the elastic connective tissue (see below). Theyhave been isolated from the human placenta and inter-acted with platelets in a similar way as aortic microfi-brils (Legrand et al. 1986). Furthermore, microfibrils,but not amorphous elastic fibres, were demonstrated inthe extracellular matrix of peripheral portions of placen-tal villi by Baccarani-Contri et al. (1989).

Our light- and electron-microscopic findings showthe presence of all components of elastic fibres in thestem villous stroma. The ultrastructure of the placentalelastic fibres generally corresponds to that of other or-gans. As shown by Cotta-Pereira et al. (1976) for theskin, the elastic fibrous system is composed of three ul-trastructurally different portions: 1) nearly homogeneouselastin, 2) delicate elaunin fibres defined as homogene-ous elastin connected to microfibrils at the outer face,and 3) oxytalan fibres which represent bundles of micro-fibrils. All these structures have been described to occurin several organs, for example, in connective tissue ofthe tunica propria of mouse bronchioles and in chickenand mouse aorta (Karrer 1958; Karrer and Cox 1960,1961), in the rat cornea (Jakus 1954) and human cardiacmuscle (Battig and Low 1961). In the present study,these three components of the elastic tissue have alsobeen shown for the human placenta, being confirmed bylight microscopy after staining of cryostat sections withacid orcein according to Taenzer-Unna (Unna 1891).This staining method was described to be specific for allthree components of the elastic tissue fibres (Romeis1989).

In our study, elastic fibres were found in differingamounts depending on their location within the PVCSpreparations. Sheath areas with rather densely packednon-ramifying smooth muscle cells and little connectivetissue were seen in contrast to loosely arranged ramify-ing cells within large amounts of connective tissue.These findings correspond to our previous light-micro-scopic results (Graf et al. 1994). The bundles of orcein-stained and α-actin-immunoreactive cells between thefoetal blood vessels correspond to the fibrous skeletonwithin stem villi as described by Tenzer (1962, see alsoBecker 1963). Tenzer graphically reconstructed thesebundles and demonstrated them to be fibrous axes locat-ed in stem villi. He, additionally, reported on fibroussheaths that surround foetal blood vessels and pointedout that these sheaths may give support to the stem villi.In his study, however, he gave no indication for smoothmuscle cells within this fibrous sheath. Our results dem-onstrate that elastic fibres, collagen fibres and smoothmuscle cells form layers within the PVCS. The follow-ing points support the supposition of the presence of afunctional myofibroelastic unit: 1) connection of thesmooth muscle cells to each other and to the elastic andcollagen connective tissue via distinct invaginations, 2)adhesion plaques with corresponding talin and fibronec-tin immunoreactivity (see below) and the 3) connectionof elastic fibres with smooth muscle cells and collagenfibres, and of collagen fibres with each other. Its struc-ture is reminiscent of the myoelastic unit shown for thetunica media of the mouse and rat aorta (Karrer and Cox

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1960, 1961; Cliff 1967), but in some respects also ofsmooth muscle cells shown in rabbit portal veins whichwere also found in close relationship with elastic fibresand microfibrils (Komuro and Burnstock 1980).

The formation of a functional myofibroelastic unitwithin the PVCS is supported by our findings of adhe-sion plaques in the extravascular smooth muscle cells,and the light-microscopic results after talin and fibronec-tin immunocytochemistry. The corresponding results oftalin and α-actin immunostaining argue strongly for thepresence of talin in placental extravascular and vascular(media) smooth muscle cells. Talin is known to be pres-ent in adhesion plaques (focal adhesions, adhaerensjunctions, cell-extracellular matrix contacts; Volk andGeiger 1984; Geiger et al. 1985). The same authors re-port a general molecular heterogeneity of adhesionplaques which may form cell-cell junctions (zonula ad-haerens junctions) and cell-extracellular matrix junc-tions. Only the cell-matrix junction contains talin. In vi-vo, adhesion plaques were found, for example, insmooth muscle cells where they may occupy the lengthof the plasmalemma (Small 1985; for references, seeBurridge et al. 1988). Dense plaques of smooth musclecells show the same protein assembly as adhesionplaques of cultured cells which consist, in addition totalin, of vinculin and α-actinin (Small 1985; Drenck-hahn et al. 1988). The function of this protein assemblyis to link α-actin to extracellular fibronectin via vinculinand α-actinin and to transmit tension across the plasmamembrane (for references, see Burridge et al. 1988). Inthe present study, the obvious linkage of collagen fibresto each other via spike-like structures, of microfibrils tocollagen fibres and of elastin to smooth muscle-like cellsmay complete the myofibroelastic unit as a functionalunit. The spike-like structures on collagen fibres may re-present so-called FACIT collagens (types IX, XII andXIV) which serve to link fibrillar collagens with otherextracellular matrix macromolecules (Olsen and Ni-nomiya 1993). A candidate of these FACIT collagensrepresenting the spike-like structures shown in the pres-ent study may be undulin, a noncollagenous extracellularmatrix protein which was isolated from the human pla-centa and skin (Schuppan et al. 1990). In their study,these authors demonstrated that undulin is associatedwith collagen type I fibres.

In summary, our investigation has shown that theperivascular contractile sheath of placental trunci andrami chorii represents a myofibroelastic unit, which maylead to elasticity in addition to tensile and/or contractileforces. Contractions of the extravascular smooth musclecells may cause stretching of the elastic fibres, whichmay be reversed by recoiling of the elastic fibres. Theinterconnection between elastic and collagen fibres maylimit the extent of stretching. The architecture of the pla-cental perivascular contractile sheaths strongly supportsthe hypothesis of Dubreuil and Rivière (1932) and Span-ner (1936) that due to the contraction of the extravascu-lar smooth muscle cells of human placental stem villi thechorionic plate may approach the basal plate. Thiswould affect blood flow in the intervillous space. Con-traction and relaxation of the PVCS in the longitudinal

direction to the stem villi could lead to propulsive forceson the adjacent foetal blood vessels. However, further in-vestigations will be necessary to prove this interestinghypothesis.

&p.2:Acknowledgements.The authors are indebted to N. Knop and D.Matejevic for skilful preparations of stem villi, to M. Gutsmann,M. Joncic and U. Sauerbier for excellent technical assistance, toDr. J.U. Langer for his technical support in electron-microscopicwork, and to B. Steyn for linguistic corrections. Helpful literaturewas obtained from the collection of the scientific literature of thelate Professor H. Lemtis. The generous gift of this collection byDr. med. I. Lemtis to one of us (R. Graf) is gratefully acknowl-edged.

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