Cell Tissue Res (1992) 270:241-248

Cell&Tissue Research © Springer-Verlag 1992

Distribution of laminin, type IV collagen, and fibronectin in the cell columns and trophoblastic shell of early macaque placentas Thomas N. Blankenship, Allen C. Enders, and Barry F. King Department of Cell Biologyand Human Anatomy, School of Medicine, University of California, Davis, Davis, CA 95616, USA ReceivedApril 20, 1992 / AcceptedJune 22, 1992

Summary. The cytotrophoblastic cell columns and trophoblastic shell of macaque placentas accumulate progressively greater amounts of intercellular material during early gestation. We studied the composition of this material in placentas collected from 22 34 days of gestation by using immunoperoxidase techniques directed to the extracellular matrix molecules fibronectin, type IV collagen, and laminin. These antigens co-localized within the intercellular deposits at all stages studied. At day 22 the proximal cell columns were composed of cells with narrow interstices and which lacked immunoreactivity for the 3 antigens. Distally the cells were vacuolated and the intercellular spaces increased in size and contained dense matrix deposits. The trophoblastic shell consisted of closely packed, non-vacuolated cytotrophoblast cells with only a delicate meshwork of matrix. By day 27 the matrix deposits of the distal cell columns increased markedly in size. The trophoblastic shell contained larger numbers of vacuolated cells and was occupied by accumulations of matrix. By 34 days the matrix deposits of the cell columns expanded substantially along the longitudinal axes of the columns. These deposits were often continuous with a matrix-dense, cell-deficient layer in the trophoblastic shell. This matrix-rich zone lay between a cellular layer adjacent to the intervillous space and a similar, but discontinuous, cell layer that formed the junctional zone with the endometrium. Key words" Trophoblast - Placenta - Laminin - Collagen - Fibronectin - Extracellular matrix,-structures Macaca fascicularis (Primates)

During development of the primate placenta, cytotrophoblastic cell columns extend from the tips of the anchoring villi to the floor of the intervillous space. At their distal ends the columns spread and merge to form a layer termed the trophoblastic shell (Wislocki and Correspondence to: B.F. King

Streeter 1938; Wislocki and Bennett 1943; Wilkin 1965; Boyd and Hamilton 1970; Benirschke and Kaufmann 1990). Morphogenetic changes in the columns and trophoblastic shell are probably important in trophoblast interactions with the endometrium and for placental growth and expansion. The cytotrophoblast cells of the columns differentiate as they extend toward the shell. Cytotrophoblast cells of the proximal portions of the cell columns are closely packed, mitotically active cells with narrow interstices. In the more distal portions of the cell columns mitotic activity is thought to be reduced and the cells become more vacuolated in routine preparations, primarily due to extraction of glycogen (Wislocki and Bennett 1943). Another important feature of the distal columns is that cytotrophoblast cells become separated by the accumulation of variable amounts of extracellular material. The origin and composition of this material has been the subject of some interest over the years, but with few definitive conclusions. Grosser (1925) [as summarized by Wislocki and Bennett 1943], thought that fibrinold in human cell columns was primarily a secretory product of the fetal trophoblast. Wislocki and Streeter (1938) noted the development of large regions of extracellular material within the cell columns of early rhesus monkey placentas, usually referring to them as fluidfilled edematous lakes. Wislocki and Bennett (1943) generally referred to this material as matrix, ground substance, or "fibrinoid" and thought of it primarily as breakdown products of maternal (decidual) origin having a role in embryonic nutrition. They referred to the origin and genesis of fibrinoid, in particular, as "one of the most obscure problems of placental histology". Nearly fifty years later, Benirschke and Kaufmann (1990) noted that "the question as to the precise nature of the fibrinoid is still open". Reviews of the history of this terminology can be found in Boyd and Hamilton (1967, 1970). In order to define better the origins and composition of the extracellular matrix (ECM) in the cell columns and trophoblastic shell of the developing primate placen-

242 ta, we have carried out an immunohistochemical study of early macaque placentas using antibodies to type IV collagen, laminin, and fibronectin. The stages examined (between 22 and 34 days of gestation) include ages studied by Wislocki and Streeter (1938) and Wislocki and Bennett (1943), enabling us to correlate our observations with these historically important papers.

Materials and methods Cynomolgus monkeys (Macaca fascicularis) were housed at the California Regional Primate Research Center. Animals were mated twice, 2 days apart, at the anticipated time of ovulation based on records of previous menstrual cycles, with the second mating designated day 0 of pregnancy. On day 16 of pregnancy the presence of a conceptus was confirmed by ultrasound diagnosis. Placentas were collected by surgical embryectomy which included removal of the entire conceptus and surrounding endometrial tissue. Placentas were examined from 4 animals collected on days 22, 27, 33, and 34 of pregnancy. Placentas were fixed by immersion in chilled phosphate-buffered 4% paraformaldehyde immediately after removal from the uterus. Tissues were rinsed overnight in buffer prior to processing for routine paraffin embedding. Sections were cut at a thickness of 6 gm and mounted on poly-L-lysine-coated slides. Following deparaffinization and rehydration, the sections were treated for 15 rain, 30 min, or 2 h in pepsin-acetic acid to expose antigenic sites (1 mg/ml pepsin in 0.5 M glacial acetic acid; Barsky et al. 1984) and rinsed in water. Each of the following steps was succeeded by thorough rinsing in phosphate-buffered saline. Endogenous peroxidase activity was blocked by 15 min incubation with 3% hydrogen peroxide in methanol. Nonspecific antibody binding was minimized by treatment with 10% normal goat serum (Gibco; Grand Island, NY) for 30 min. Rabbit antibodies (and dilution) to laminiu (1:250, E-Y Laboratories; San Mateo, Calif.), fibronectin (1:1500, E-Y Laboratories; San Mateo, Calif.), and type IV collagen (1:2000, Chemicon; Temecula, Calif.) were applied for 2 h. Sections were then incubated in goat-antirabbit IgG antibodies (1:50, Cappel; Organon Teknika Corp., Durham, NC) for 30 min. Finally, rabbit antiperoxidase antibodies conjugated to peroxidase (1:500, Cappel; Organon Teknika Corp., Durham, NC) were applied for 30 min. The presence of bound peroxidase was indicated by development in chromogen (7.5 mg diaminobenzidine tetrachloride in 10 ml of 0.05 M Tris buffer with 20 gl 30% hydrogen peroxide) for 3-5 min. Sections were counterstained in hematoxylin. Controls consisted of replacing the usual primary antibodies with antibody preadsorbed with the appropriate antigen. In addition, normal rabbit serum was substituted for the primary antibody. In each case, immunostaining on tissue sections was nearly abolished. Tissues from primary and secondary placentas were examined. No differences in the distribution of ECM were found between these tissues from the same animal, therefore no further distinction will be made in this regard.

Results The basic features of the early placenta were apparent at 22 days of gestation (Fig. 1). Stem villi emanated from the chorionic plate and gave rise to a few free villous branches. Anchoring villi were numerous and, at their distal tips, continued as the cytotrophoblastic cell columns. As the cell columns extended distally (toward the maternal decidua) they spread laterally and merged to form the trophoblastic shell. The boundary between the

trophoblastic shell and the decidua was quite even. The most proximal portions of the cell columns (near the stroma of anchoring villi) consisted of densely-packed cells, but were essentially devoid of immunoreactive type IV collagen, laminin, and fibronectin deposits (Fig. 2). A few cells more distally, however, immunoreactive deposits were observed ifitracellularly as well as between the cells. The cytotrophoblast cells became vacuolated in the distal columns and, concomitantly, E C M deposits were increasingly prominent between the cells (Figs. 1, 2). Laminin, fibronectin, and type IV collagen all appeared to be co-localized in these regions. Most of the trophoblastic shell at this stage consisted of closelypacked, non-vacuolated cytotrophoblast and only a sparse meshwork of pericellular ECM was found in this zone. By 27 days of gestation, the area occupied by each of the ECM components had increased substantially. Particularly notable were the large deposits or " l a k e s " of E C M in the distal cell columns (Figs. 3-6). These accumulations came to occupy expansive regions throughout the cell columns (Figs. 4-6), although the proximal areas remained free of immunoreactivity. The trophoblastic shell, which became increasingly populated by vacuolated trophoblast cells during this time, also showed larger deposits of ECM, particularly in the proximal layers of the shell (Figs. 3, 4, 6). At all gestational stages examined the junctional zone of the placenta and endometrium was sometimes interrupted by discontinuous masses of degenerating cells (Fig. 6). These masses were variable in size, appeared to be accumulations of extravasated maternal blood, and were immunoreactive for fibronectin, but not for laminin or type IV collagen. At 33-34 days of gestation, the cell columns were similar to those described at 27 days, except that the lakes of E C M in the distal regions were even more expansive, extending along the longitudinal axes of the cell columns (Figs. 7, 8). These lakes contained small, dense, granules that were especially apparent in tissue immunostained for fibronectin (Fig. 9). The particulates were also visible, though to a lesser degree, in fibronectin-stained tissue from the earlier stages and in sections stained for laminin, type IV collagen, and in non-immunostained sections (not shown). Changes had occurred in the distribution of ECM materials of the trophoblastic shell as well. The trophoblastic shell had a zone nearer the base of the intervillous space consisting of mostly vacuolated cytotrophoblast cells with sparse pericellular ECM deposits (Figs. 7, 9). Immediately distal to this zone was another layer or zone that appeared less densely populated by cells and contained more E C M which was strongly immunoreactive for type IV collagen, laminin, and fibronectin (Figs. 7, 9). The interface of this densely reactive layer with more distally located components of the trophoblastic shell was variable in contour. In some cases, a thin layer of cytotrophoblast with sparse ECM deposits was observed (Figs. 7, 9). In other cases, fibronectin-positive areas of necrotic tissue were located at the interface with maternal tissue. For all of the stages studied, the endometrium sur-

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Fig. 1. Low-magnification micrograph of entire thickness of macaque placenta, 22 days of gestation, immunostained for laminin. Stem villi emerge from chorionic plate (CP), but villi are relatively unbranched at this age. Cytotrophoblastic cell columns (CC) merge distally to form trophoblastic shell (TS). Although difficult to see at this magnification, laminin is localized predominantly in distal cell columns (arrows). x 40

man endometrium (Wewer et al. 1985). Cytotrophoblast cells invaded or displaced portions of the walls of uterine veins. In these areas the endothelial basement membrane was lost, although basement membrane subtending adjacent hypertrophied venous endothelium remained intact (Figs. 3, 10). The duration of pepsin digestion was an important factor regarding the appearance of the E C M lakes. Immunolabeling of type IV collagen and fibronectin was clearly defined in placental and most maternal tissues following 15 rain digestion. Laminin labeling was enhanced by 30 rain exposure to the pepsin. Following 2 h incubation in pepsin the basement membranes of fetal and uterine tissues were clearly stained (not shown). However, the matrix lakes of the cell columns were far less immunoreactive and, in some cases, completely vacant, suggesting that prolonged digestion removed matrix antigens from these areas.

Discussion

Fig. 2. Higher magnification of 22-day placenta stained for fibronectin. Prominent labeling is seen around trophoblast cells in distal cell columns (arrows), but not proximally. Fetal connective tissue (F) in anchoring villi and maternal decidua (D) are also immunoreactive; TS trophoblastic shell, x 90

rounding the trophoblastic shell stained for laminin and type IV collagen in the basement membranes surrounding blood vessels and uterine glands. Localization o f fibronectin along basement membranes was inconsistent and usually weak. The decidual cells were surrounded by boundary laminae that were also immunoreactive for each of the antigens examined (e.g., type IV collagen, Fig. 10), consistent with results of earlier work on hu-

Our results have demonstrated the accumulation of large amounts of basement membrane-related molecules in the cytotrophoblastic cell columns and trophoblastic shell of early macaque placentas. Furthermore, our observations indicate that the type IV collagen, laminin, and fibronectin in the distal cell columns were products of the cytotrophoblast. The latter conclusion is based on the demonstration of cytoplasmic staining for the antigens in the cytotrophoblast cells as well as the fact that cytotrophoblast was the only cell type present in the regions of greatest accumulation. These results are consistent with other reports localizing these antigens in early (usually 8-12 week) human placentas (Yamaguchi etal. 1985; Yamada et al. /987; Earl et al. 1990; Damsky et al. 1992) and results of a study of cell columns utilizing in situ hybridization which showed that cells in the columns expressed both laminin and type IV collagen m R N A (Autio-Harmainen et al. 1991). Feinberg et al. (1991) have localized a particular class o f fibronectin, oncofetal fibronectin, in the cell columns.

Fig. 3. Low-magnification micrograph of placenta at 27 days of gestation stained for type IV collagen. Prominent staining is present in fetal villous stroma (F) and distal cell columns (arrows). Less intense staining is seen between cells of trophoblastic shell (TS). Portions of uterine vein basement membrane (arrowheads) are lost where cytotrophoblast cells (CT) have invaded and replaced vessel wall. x 40

Fig. 4. Higher magnification of type IV collagen localization in 27 day placenta. Scattered deposits of type IV collagen are seen between many cells in proximal cell columns (arrows), but staining becomes much more pronounced in distal cell columns. Pericellular staining is also seen in trophoblastic shell (TS). x 90

Fig. 5. Twenty-seven day placenta stained for laminin. Prominent staining is apparent in distal cell columns, but is weak in proximal portion of columns (CC) and trophoblastic shell (TS). x 90

Fig. 6. Twenty-seven day placenta stained for fibronectin. Pericellular staining is weak in proximal cell columns (CC), but progressively increasing fibronectin deposits are seen in distal columns. Delicate intercellular staining is present in trophoblastic shell (TS) and decidua (D). A small mass of fibronectin-positive deteriorating cells is apparent near junction of trophoblastic shell with decidua (arrow). x 90

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Fig. 7. Thirty-three day placenta stained for type IV collagen. As was the case with other antigens studied, delicate deposits are found in proximal cell columns (CC), whereas abundant deposits are located in distal columns. At this stage, in the portion of trophoblastic (TS) shell nearest the intervillous space there is a cellular zone characterized by pericellular collagen deposits. The central zone of the shell has fewer cells, but extensive deposits of ECM. Distally, the shell has more of a reticulated intercellular staining pattern. Decidual cells (D) are surrounding by boundary laminae, x 90

Fig. 8. Cell columns (CC and trophoblastic shell (TS) of 33-day placenta stained for fibronectin. Distally, increasingly extensive deposits occupy much area of the cell columns. Pericellular staining of variable intensity is also seen around trophoblast cells in the shell, x 100

Fig. 9. Higher magnification of junction of distal cell column with trophoblastic shell, stained for fibronectin. Within extensive ECM deposits in distal columns, intensely stained granules are seen (arrowheads). Pericellular staining is seen around some trophoblast cells in trophoblastic shell (TS). Note 3 strata typical of trophoblastic shell at this stage : inner layer, containing vacuolated cells, adjacent to intervillous space (1); central zone rich in ECM (2); and outer cell layer with pericellular ECM (3). 33 days, x 250

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Fig. 10. Cytotrophoblast cells (Cir) have invaded an endometrial vein and replaced a portion of its wall. In this region, endothelial basement membrane is lost although it remains visible on opposite side of vessel (arrow). Type IV collagen-positivecapsules are present around decidual cells (arrowheads). Trophoblastic shell TS; cytotrophoblasticcell column CC. 33 days, x 180 Thus there is good correspondence between our observations on these very early macaque placentas and observations of human placentas from somewhat later in gestation. By identifying cytotrophoblast as a primary source of type IV collagen, laminin and fibronectin in the cell columns, we have confirmed Grosser's (1925) original speculation as to the origin o f " fibrinoid" material in this region of the human placenta. These results are also consistent with results from in vitro studies that demonstrate that cytotrophoblast cells isolated from human placentas synthesize and secrete fibronectin, oncofetal fibronectin, laminin, and type IV collagen (UlloaAguirre etal. 1987; Yagel etal. 1988; Kliman etal. 1989; Feinberg et al. 1991 ; Damsky et al. 1992). At the present time we can only speculate as to the functional roles of the prominent ECM accumulations in the distal cell columns. Since the expansive regions of ECM accumulation are in the direction of the trophoblastic shell, one hypothesis is that the matrix has a role in facilitating trophoblast migration relating to expansion and remodeling of the cell columns and trophoblastic shell. The synthesis and deposition of large amounts of laminin, fibronectin and type IV collagen in the distal cell columns may promote trophoblast cell

adhesion to these ECM molecules as well as haptotactic cell migration from the proximal portion of the cell columns to the distal columns and trophoblastic shell. Certain embryonic cells (Linask and Lash 1986) and tumor cells show a haptotactic response to fibronectin and/or laminin (McCarthy and Furcht 1984; McCarthy et al. 1986; Furcht et al. 1984; Wewer et al. 1987). Fibronectin, type IV collagen (Kao et al. 1988), and laminin (Loke et al. 1989) appear to be involved in human trophoblast attachment in vitro, and mouse trophoblast cells also adhere to fibronectin, laminin, and collagen (Armant et al. 1986; Carson et al. 1988; Sutherland et al. 1988). Migration may be enhanced by the presence of ECM receptors on trophoblast cells. Kao et al. (1988) suggested the presence of fibronectin receptors on human trophoblast cells in vitro, and invading trophoblast cells of early human placentas express a laminin receptor (Wewer et al. 1987). The presence of integrin-type receptors on trophoblast cells has been reported recently. Korhonen etal. (1991) examined human placentas and found that migratory intermediate trophoblast was immunoreactive for the integrin subunits /~, ez, c%, and c~5. Spatiotemporal shifts in the distribution of integrins were found in the cytotrophoblastic cell columns of human placentas (Damsky et al. 1992). The % and/3~ subunits were abundant in the proximal columns while ~5 and 1~1 were prominent in the distal columns. These integrin changes were concomitant with the accumulation of ECM in the distal column. The results of these latter 2 reports indicate that the integrins found on trophoblast are sufficient for adhesion to ECM proteins known to be present in the cell columns and trophoblastic shell (Ruoslahti 1991). There are few comparable examples of extended basement membrane-like regions during embryonic development. One area with some similarities is the cardiac jelly of the embryonic heart which has been likened to an extended basement membrane (Kitten et al. 1987; Little et al. 1989). However, the distal cell columns of the placenta have a much broader, homogeneous, distribution of type IV collagen, fibronectin, and laminin than cardiac jelly. One interesting similarity between the two regions is the finding of particulate components in the ECM. In cardiac jelly these particulates occur in a gradient and consist of fibronectin and a variety of glycoproteins (Mjaatvedt et al. 1987; Sinning et al. 1992). The particles we observed stained prominently for fibronectin, but it was unclear whether, in the case of the placenta, the particles are arranged in a gradient. Laminin and type IV collagen have also been found distributed as granules, not incorporated into basement membranes, in other developing tissues (Rogers et al. 1986; Riggott and Moody 1987; Solursh and Jensen 1988; Zhou 1990). Therefore, as suggested by Little et al. (1989), the view that limits type IV collagen and laminin distribution to basement membranes may be too restricted when applied to embryonic tissues. Movement of the cells distally may involve the activity of ECM metalloproteinases (Woessner 1991). Fernandez et al. (1992) demonstrated, in vivo, the presence of type IV collagenase in human first trimester cytotrophoblastic cell columns. Early human embryos express

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matrix degrading proteolytic activity in vitro (Puistola et al. 1989). Trophoblast cells isolated from first trimester human placentas have several metalloproteinases (Fisher et al. 1989; Bischof et al. 1991; Librach et al. 1991) including collagenase (Yagel et al. 1988; Emonard etal. 1990; Moll and Lane 1990; Graham and Lala 1991). Human trophoblast cells also produce plasminogen activator (Queenan et al. 1987; Yagel et al. 1990) as well as plasminogen activator inhibitors (Feinberg et al. 1989). These enzymes have been implicated in trophoblastic invasion of the decidua, and it is possible that they are active in ECM degradation in the regions we describe here. The observation that cells from the distal portion of the trophoblastic shell apparently degrade portions of the basement membrane as they invade and replace portions of the walls of the uteroplacental veins suggests that at least this subpopulation of cells possesses matrix-degrading capacity. Another possible function of the elaborate ECM produced in the distal cell columns may be to modulate trophoblastic differentiation pathways and cell function. Attachment of human cytotrophoblast to fibronectincoated or type IV collagen-coated matrices in vitro promotes syncytium formation (Kao et al. 1988) whereas cytotrophoblast cells plated onto complex ECM (Matrigel) tend to remain mononuclear and demonstrate invasive behavior (Kliman and Feinberg 1990; Librach et al. 1991). Laminin is also known to have effects on cell proliferation and differentiation (reviewed by Paulsson 1992). Regional modifications of cytotrophoblast integrins occur in concert with ECM deposition in the cell columns of human placentas (Damsky et al. 1992). Feinberg et al. (1991) suggested that the oncofetal fibronectin they localized in distal cell columns may function as a "trophouteronectin" for implantation and placental anchorage to the uterine wall. Our results demonstrate that there are additional ECM molecules that are abundant in this region of the macaque placenta. Whether type IV collagen, laminin, and/or fibronectin also function in this manner is equally speculative. The origin of the deteriorating masses in the junctional zone is uncertain. Many of them had an overall circular or elliptical shape. This, coupled with apparent blood cell content, suggest some of them may be the result of occluded maternal blood vessels or extravasated blood. This condition could occur following invasion of an endometrial vessel that does not open directly into the intervillous space. The positive staining for fibronectin is consistent with this origin. Other degenerating areas may be the result of epithelial plaque cells or decidua. Autio-Harmainen et al. (1991) thought that the basement membrane material they identified in cell columns of early human placentas was important in the formation of future villous stroma. Apparently this idea was based on their erroneous assumption that "new villi are formed from the cells of the cytotrophoblastic columns". An abundance of evidence clearly indicates that this is not the case (summarized in Benirschke and Kaufmann 1990). In the macaque placenta, we have previously shown that during the stages of development studied here, free villous branching is mainly occurring nearer the chorionic plate, not in the cell column regions (King

and Mais 1982). Furthermore, the ECM of the columns accumulates distally, i.e., toward the endometrium and not in the direction of the chorionic plate. The ECM deposits of the cell columns were more sensitive to the effects of the pepsin incubation than were the basement membranes in the placenta and endometrium. This may reflect quantitative and qualitative differences in the molecular organization of the various ECMs encountered in these tissues (Leu and Damjanov 1988; Sanes et al. 1990).

Acknowledgments. This study was supported by NIH grants HD 24491 (B.F.K., A.C.E.), RR 00169 and a Merck Academic Development Program Postdoctoral Fellowship to T.N.B. The authors especially thank Dr. A.G. Hendrickx, Pam Peterson, and the staff of the California Regional Primate Research Center for their assistance in collecting tissues.

References Armant DR, Kaplan HA, Lennarz WJ (1986) Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts. Dev Biol 116:519-523 Autio-Harmainen H, Sandberg M, Pihlajaniemi T, Vuorio E (I 991) Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta. Lab Invest 64:483 491 Barsky SH, Rao NC, Restrepo C, Liotta L (1984) Immunocytochemical enhancement of basement membrane antigens by pepsin: applications in diagnostic pathology. Am J Clin Pathol 82:191-194 Benirschke K, Kaufmann P (1990) Pathology of the Human Placenta, 2nd edn. Springer, New York Bischof P, Friedli E, Martelli M, Campana A (1991) Expression of extracellular matrix-degrading metalloproteinases by cultured human cytotrophoblast cells: effects of cell adhesion and immunopurification. Am J Obstet Gynecol 165:1791-1801 Boyd JD, Hamilton WJ (1967) Development and structure of the human placenta from the end of the 3rd month of gestation. J Obstet Gynaecol Br Commonw 74:161-226 Boyd JD, Hamilton WJ (1970) The Human Placenta, Heifer, Cambridge Carson DD, Tang J-P, Gay S (1988) Collagens support embryo attachment and outgrowth in vitro: effects of the arg-gly-asp sequence. Dev Biol 127:368 375 Damsky CH, Fitzgerald ML, Fisher SJ (1992) Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo. J Clin Invest 89 : 210-222 Earl U, Estlin C, Bulmer JN (1990) Fibronectin and laminin in the early human placenta. Placenta 11:223-231 Emonard H, Christiane Y, Smet M, Grimaud JA, Foidart JM (1990) Type IV and interstitial collagenolytic activities in normal and malignant trophoblastic cells are specifically regulated by the extracellular matrix. Invas Metast 10:170-177 Feinberg RF, Kao L-C, Haimowitz JE, Queenan JT, Wun T-C, Strauss III JF, Kliman HJ (1989) Plasminogen activator inhibitor types 1 and 2 in human trophoblasts. Lab Invest 61:20-26 Feinberg RF, Kliman HJ, Lockwood CJ (1991) Is oncofetal fibronectin a trophoblast glue for human implantation ? Am J Pathol 138:537-543 Fernandez PL, Merino M J, Nogales FF, Charonis AS, StetlerStevenson W, Liotta L (1992) Immunohistochemical profile of basement membrane proteins and 72 kilodalton type IV collagenase in the implantation placental site. Lab Invest 66:572579 Fisher SJ, Cui T-Y, Zhang L, Hartman L, Grahl K, Gou-Yang Z, Tarpey J, Damsky CH (1989) Adhesive and degradative

248 properties of human placental cytotrophoblast cells in vitro. J Cell Biol 109 : 891-902 Furcht LT, McCarthy JB, Palm SL, Basara ML, Enenstein J (1984) Peptide fragments of laminin and fibronectin promote migration (haptotaxis and chemotaxis) of metastatic cells. In: Porter R, Whelen J (eds) Basement Membranes and Cell Movement, Ciba Foundation Symposium No. 108, Pitman, London, pp 130-145 Graham CH, Lala PK (1991) Mechanism of control of trophoblast invasion in situ. J Cell Physiol 148:228-234 Grosser O (1925) Uber Fibrinoid in der Placenta. Zeitschr Anat Entw 76:304-314 Kao L-C, Caltabiano S, Wu S, Strauss III JF, Kliman HJ (1988) The human villous cytotrophoblast: interactions with extracellular matrix proteins, endocrine function, and cytoplasmic differentiation in the absence of syncytium formation. Dev Biol 130: 693-702 King BF, Mais JJ (1982) Development changes in rhesus monkey placental villi and cell columns. Anat Embryol 165:361-376 Kitten GT, Markwald RR, Bolender DL (1987) Distribution of basement membrane antigens in cryopreserved early embryonic hearts. Anat Rec 217:379-390 Kliman HJ, Feinberg RF (1990) Human trophoblast-extracellular matrix (ECM) interactions in vitro: ECM thickness modulates morphology and proteolytic activity. Proc Natl Acad Sci USA 87:3057-3061 Kliman H J, Coutifaris C, Feinberg RF, Strauss III JF, Haimowitz JE (1989) Implantation: in vitro models utilizing human tissues. In: Yoshinaga K (ed) Blastocyst Implantation. Adams, Boston, pp 83-91 Korhonen M, Ylfinne J, Laitinen L, Cooper HM, Quaranta V, Virtanen I (1991) Distribution of the cq-c% integrin subunits in human developing and term placenta. Lab Invest 65:347-356 Leu F-J, Damjanov I (1988) Protease treatment combined with immunohistochemistry reveals heterogeneity of normal and neoplastic basement membranes. J Histochem Cytochem 36:213-220 Librach CL, Werb Z, Fitzgerald ML, Chiu K, Corwin NM, Esteves RA, Grobelny D, Galardy R, Damsky CH, Fisher SJ (1991) 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol 113:437-449 Linask KK, Lash JW (1986) Precardiac cell migration: fibroneetin localization at mesoderm-endoderm interface during directional movement. Dev Biol 114:87-101 Little CD, Piquet DM, Davis LA, Walters L, Drake CJ (1989) Distribution of laminin, collagen type IV, collagen type I, and fibronectin in chicken cardiac jelly/basement membrane. Anat Rec 224:417-425 Loke YW, Gardner L, Burland K, King A (1989) Laminin in human trophoblast-decidua interaction. Human Reprod 4:457463 McCarthy JB, Furcht LT (1984) Laminin and fibronectin promote the haptotactic migration of B16 mouse melanoma cells in vitro. J Cell Biol 98 : 1474-1480 McCarthy JB, Hagen ST, Furcht LT (1986) Human fibronectin contains distinct adhesion- and motility-producing domains for metastatic melanoma cells. J Cell Biol 102:179-188 Mjaatvedt CH, Lepera RC, Markwald RR (1987) Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin. Dev Biol 119 : 59-67 Moll UM, Lane BL (1990) Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast. Histochemistry 94:555-560 Paulsson M (1992) Basement membrane proteins: structure, assembly, and cellular interactions. Crit Rev Biochem Molec Biol 27:93-127 Puistola U, R6nnberg L, Martikainen H, Turpeenniemi-Hujanen T (1989) The human embryo produces basement membrane collagen (type IV collagen)-degrading protease activity. Human Reprod 4: 309-31

Queenan JT, Kao L-C, Arboleda CE, Ulloa-Aguirre A, Golos TG, Cines DB, Strauss JF III (1987) Regulation of urokinase-type plasminogen activator production by cultured human cytotrophoblasts. J Biol Chem 262:10903-10906 Riggot M J, Moody SA (1987) Distribution of laminin and fibronectin along peripheral trigeminal axon pathways in the developing chick. J Comp Neurol 258: 580-596 Rogers SL, Edson KJ, Letourneau PC, McLoon SC (1986) Distribution of laminin in the developing peripheral nervous system of the chick. Dev Biol 113:429-435 Ruoslahti E (1991) Integrins as receptors for extracellular matrix. In: Hay ED (ed) Cell Biology of Extracellular Matrix. Plenum, New York, London, pp 343-363 Sanes JR, Engvall E, Butkowski R, Hunter DD (1990) Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J Cell Biol 111:1685-1699 Sinning AR, Krug EL, Markwald RR (1992) Multiple glycoproteins localize to a particulate form of extracellular matrix in regions of the embryonic heart where endothelial cells transform into mesenchyme. Anat Rec 232:285 292 Solursh M, Jensen KL (1988) The accumulation of basement membrane components during the onset of chondrogenesis and myogenesis in the chick wing bud. Development 104:41-49 Sutherland AE, Calarco PG, Damsky CH (1988) Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth. J Cell Biol i06:13311348 Ulloa-Aguirre A, August AM, Golos TG, Kao L-C, Sukuragi N, Kliman HJ, Strauss III JF (1987) 8-Bromo-adenosine 3',5'monophosphate regulates expression of chorionic gonadotropin and fibronectin in human cytotrophoblasts. J Clin Endocrinol Metab 64:100~1009 Wewer UM, Faber M, Liotta LA, Albrechtsen R (1985) Immunochemical and ultrastructural assessment of the nature of the pericellular basement membrane of human decidual cells. Lab Invest 53:624-633 Wewer UM, Taraboletti G, Sobel ME, Albrechtsen R, Liotta LA (1987) Role of laminin receptor in tumor cell migration. Cancer Res 47 : 5691-5698 Wilkin PG (1965) Organogenesis of the human placenta. In: DeHaan RL, Ursprung H (eds) Organogenesis. Holt, Rinehart and Winston, New York, pp 743-769 Wislocki GB, Bennett HS (1943) The histology and cytology of the human and monkey placenta, with special reference to the trophoblast. Am J Anat 73:335-449 Wislocki GB, Streeter GL (1938) On the placentation of the macaque (Macaca mulatta), from the time of implantation until the formation of the definitive placenta. Contrib Embryo127:166 Woessner JF (1991) Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5:2145-2154 Yagel S, Parhar RS, Jeffrey JJ, Lala PK (1988) Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells. J Cell Physiol 136: 455-462 Yagel S, Kerbel R, Lala P, Eldar-Gera T, Dennis JW (1990) Basement membrane invasion by first trimester human trophoblast: requirement for branched complex-type Asn-linked oligosaccharides. Clin Exp Metast 8:305-317 Yamada T, Isemura M, Yamaguchi Y, Munakata H, Hayashi N, Kyogoku M (1987) Immunohistochemical localization of fibronectin in the human placentas at their different stages of maturation. Histochemistry 86: 579-584 Yamaguchi Y, Isemura M, Yosizawa Z, Kurosawa K, Yoshinaga K, Sato A, Suzuki M (1985) Changes in the distribution of fibronectin in the placenta during normal human pregnancy. Am J Obstet Gynecol 152: 715-718 Zhou FC (1990) Four patterns of laminin-immunoreactive structure in developing rat brain. Dev Brain Res 55:191-201