ARTICLE |
Correspondence to: Matti Korhonen, Hospital for Children and Adolescents, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland. E-mail: matti.korhonen@helsinki.fi
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Summary |
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We studied the localization of laminin 1,
2,
3,
5,
1,
2, and
1 chains and extradomain A- (EDA), EDB-, and oncofetal fibronectin by immunohistochemistry in human placental villi during placental development. The laminin
2,
5,
1,
2, and
1 chains were detected in the trophoblastic basement membrane (BM) at all stages of gestation, suggesting the presence of laminin-2, -4, -10, and -11 trimers. The laminin
1 chain was selectively found at sites where the villous BM was in contact with proliferating cells in trophoblastic islands or columns. EDA-Fn, but not other Fn isoforms, was found in the trophoblastic BM during the first trimester. The laminin
2,
1,
2, and
1 chains were detected in the villous stroma and capillaries throughout placental development, while the laminin
5 chain emerged distinctly during development. Extensive EDA-Fn immunoreactivity was found in first-trimester villous stroma, but distinctly fewer Fn isoforms were seen in the villous stroma during the later stages of gestation. Our results also suggest that, during the formation of new villi, laminins are not found in trophoblastic sprouts before the ingrowth of the villous mesenchyme. Rather, laminins may be deposited at the villous epithelialmesenchymal interface. Furthermore, the results show that distinct changes occur in the localization of various laminin and Fn isoforms during the maturation of villous trophoblastic and capillary BMs. (J Histochem Cytochem 49:313322, 2001)
Key Words: placenta, laminin, fibronectin, basement membrane, extracellular matrix proteins, immunohistochemistry, human
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Introduction |
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Basement membranes (BMs) are specialized extracellular matrices found subjacent to epithelial sheets. In addition to contributing to the structural organization of epithelial tissue, BMs are essential to the development and maintenance of epithelial cell differentiation (
Laminins are important constituents of BMs. Since the first isolation of laminin-1 from a murine tumor (-,
-, and a
-type chain, where different chain isoforms may form heterotrimers rather promiscuously (
Fibronectin is a ubiquitous component of the extracellular matrix (ECM). Fibronectin is found as an intrinsic component in some BMs during development (
Earlier, we have studied the distribution of integrins and ECM molecules in the developing and term human placenta (
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Materials and Methods |
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Tissues
Sixteen human placentas (seven 810 weeks, five 1220 weeks, and four 3840 weeks of gestation) were aquired from spontaneous abortions due to rupture of fetal membranes, from legal abortions performed for psychosocial indications, or from normal deliveries at the Jorvi Hospital, Espoo, Finland. The tissues were frozen in liquid nitrogen and stored at -70C until use.
Antibodies
The following MAbs and antisera to laminin and fibronectin isoforms were used in the study: 1 (clone EB7,
2 (clone 5H2,
3 (clone BM-2,
5 (clone 4C7,
1 (clone 4E10,
2 (clone C4,
3 (clone 6F12,
1 (clone 2E8,
Immunohistochemistry and Microscopy
Five-µm frozen sections were fixed in acetone at -20C for 10 min. For indirect immunofluorescence microscopy, the sections were incubated with the primary antibody and subsequently with the secondary antibodies (fluorescein isothiocyanatecoupled goat anti-mouse IgG and tetramethylrhodamine-coupled goat anti-rabbit IgG sera; Jackson Immunoresearch, West Grove, CA) at room temperature for 30 min. Negative controls, in which sections were exposed only to the secondary antisera, did not display unspecific immunoreactivity. The specimens were embedded in sodium-veronal/glycerol buffer (1:1; pH 8.4), or Mowiol in the case of lectins, and were examined with a Leitz Aristoplan microscope equipped with appropriate filters.
The distribution of the various laminin and fibronectin chains was evaluated by indirect immunofluorescence from sets of serial sections of the placental samples. Histology of the immunofluorescence sections was evaluated by light microscopy of hematoxylineosin-stained adjacent sections. The nomenclature for placental structures has been adapted from
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Results |
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The results are summarized in Table 1. For each aspect mentioned in the table, sections from at least three placental tissue blocks were studied. The immunofluorescence results on different tissue samples were consistent, with no major variations in fluorescence intensity or distribution.
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The Villous Trophoblastic BM
Antibodies detecting laminin 1 (Fig 1a and Fig 1b),
5 (Fig 1c), and
2 (Fig 1d) chains reacted avidly in a linear pattern at the periphery of placental villi of all three trimesters. Double immunostainings using the anti-cytokeratin serum (Fig 1a and Fig 1b), identifying the trophoblast, indicated that these laminin chains were found at the basal aspect of the villous trophoblast, apparently in trophoblastic BMs. Furthermore, a weaker but convincing BM-like immunoreactivity for
2 (Fig 1e) and
1 (Fig 1f) chains was detected basally to the villous trophoblast. No distinct differences were noted in the distribution of laminin chain immunoreactivities in trophoblastic BMs from placentas at various stages of gestation.
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Interestingly, distinct immunoreactivity for the laminin 1 chain was localized selectively in trophoblastic BMs at sites of formation of extravillous trophoblast. In first-trimester placentas, when extravillous trophoblast is typically found in trophoblastic cell islands, immunoreactivity for the laminin
1 chain was detected in the trophoblastic BM that is in direct contact with extravillous trophoblastic cells (Fig 2a and Fig 2b). The rest of the villous trophoblastic BM was negative. In second-trimester placentas, extravillous trophoblast often forms anchoring trophoblastic cell columns. Analogously to first, trimester placentas, the laminin
1 chain MAb reacted avidly with the trophoblastic BM at these sites (Fig 2c2f), whereas reactivity was not detected in BMs in other parts of villi. No immunoreactivity for the laminin
1 chain was found among the extravillous trophoblastic cells themselves within cell islands or columns. In third-trimester placentas, additional weak and irregular laminin
1 immunoreactivity was detected in the trophoblastic BM of large stem villi (not shown).
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The fibronectin and EDAfibronectin (Fig 3a) MAbs revealed linear immunoreactivity in the trophoblastic BMs of first-trimester placentas. However, these antigens were not predominantly detected in second- (Fig 3b) and third-trimester villous trophoblastic BMs. The EDBfibronectin (Fig 3c and Fig 3d) and oncofetal fibronectin (not shown) MAbs did not react with villous trophoblastic BMs.
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Villous Stroma and Capillaries
In addition to immunoreactivity in trophoblastic BMs, distinct immunoreactivity for laminin 2 (Fig 1d) and
1 (Fig 1a) chains was found in villous stroma and capillaries. The laminin
2 (Fig 1e) and
1 (Fig 1f) chain MAbs also reacted weakly with these structures. In contrast to the
2-,
1-,
2-, and
1 chain MAbs, laminin
5 chain antibodies failed to react with the villous stroma and capillaries (Fig 4a4d) of first-trimester placentas. However, intense immunoreactivity for the laminin
5 chain was detected in BMs of villous capillaries in third-trimester placentas (Fig 1c).
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The MAbs recognizing fibronectin and EDAfibronectin revealed distinct, spotty immunoreactivity in first-trimester villous stromal tissue and reacted with villous capillaries (Fig 3a). The EDBfibronectin (Fig 3c and Fig 3d) and oncofetal fibronectin MAbs reacted similarly but more weakly. In second- and third-trimester placentas, the anti-EDAfibronectin MAb (Fig 3b) reacted weakly with the villous stroma and distinctly with capillaries, while weak immunoreactivity for EDB and oncofetal fibronectin was seen in villous capillaries (not shown).
Trophoblastic Sprouts
Trophoblastic sprouts are outgrowths from the surfaces of existing villi, believed to represent sites of formation of new ones. The presence of laminin isoforms in the sprouts was determined from serial sections of first-trimester placentas. The trophoblastic sprouts were devoid of laminin and fibronectin chain immunoreactivity, as assayed by the MAbs (Fig 5a5d) as well as the EHSlaminin antiserum (Fig 5e). However, when a mesenchymal core, identified by double immu-nostaining with desmin (Fig 5c) or vimentin (not shown), was present in the sprouts defining them as villous sprouts, distinct immunoreactivity for the laminin 5,
2,
1 (Fig 5a), and weaker for
2 and
1 (Fig 5d) chains was detected in the sprouts at the trophoblastmesenchyme interphase and in the mesenchymal core.
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We also asked whether the BM at the tip of the mesenchymal villous core ingrowth into the trophoblastic villus, which may represent a site of formation of nascent BM, differs in laminin composition from the rest of the trophoblastic BM. The distributions of the immunoreactions of each laminin chain at these locations were found to be identical with that of the polyclonal EHSlaminin antiserum (Fig 5d and Fig 5e). This indicates that all laminin chains are found at the tip of the mesenchymal villous core ingrowth into the trophoblastic villus.
No immunoreactivity for the the 3 (Fig 5f) and
3 laminin chains was detected in placental villous BM or stroma at any gestational age.
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Discussion |
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The placenta and cells derived therefrom have been a major source of human laminins for biochemical studies. Earlier studies have shown that laminin is found in BMs of placental villi as well as decidual cells (2 chain is found in trophoblastic BMs. Likewise, it has been reported that fibronectin (
Antibodies
The specificity of the antibodies used in our study requires comment. The MAb C4 (anti-laminin 2) has been reported to crossreact weakly with the laminin
1 chain and with an unidentified high molecular weight band in rat tissue extracts (
2 chain in human placental tissue (
The MAb 4C7, formerly believed to recognize the laminin 1 chain, has been shown to recognize laminin
5 (
The Trophoblastic BM
In this work we demonstrate that the 2,
5,
1,
2, and
1 laminin chains are found in the villous trophoblastic BM as well as in the stroma. In agreement with these results, laminin-2, -4, -10, and -11 (
2 and
5 chains, as well as the
1 and
2 chains, have mutually exclusive distributions in BMs of various locations (
Interestingly, laminin 1 chain was found selectively in trophoblastic BMs adjacent to trophoblastic cell islands and columns. A similar restricted distribution has been described for tenascin (
1 chain at these sites is unknown at present. These results support the hypothesis that cell islands and columns are homologous structures (
5,
1, and
1 chains are found both in the trophoblastic BM and within the matrix deposition zone of anchoring cell columns, whereas the
1,
2 and
2 chains are confined to the trophoblastic BM.
1 and
2 chains would have mutually exclusive localizations. At these sites, however,
1 and
2 are co-expressed, possibly allowing the presence of laminin-3. Indeed, we have recently purified laminin-3 from human placental tissue (
Furthermore, the study shows that laminin 3 and
3 chains are not found in placental tissue, suggesting that laminins-5, -6, and -7 are not present in placenta.
Earlier studies have shown that fibronectin is found in trophoblastic BMs during the first trimester but disappears later in development (
Trophoblastic Sprouts
1 and collagen Type IV mRNA and proteins in "cytotrophoblastic columns," and hypothesized that the presence of these proteins is required for the ingrowth of the villous stroma during development of new villi. These results are somewhat contradictory to those presented in this study. In part, this is due to the general confusion of terminology in the field of placental histology. Villous trophoblastic sprouts are believed to represent sites of production of new villi from existing villi (
We also asked whether there would be differences in the distribution of the individual laminin chains at the tip of the mesenchymal ingrowth into the developing villus. We hypothesized that a differential distribution would indicate that particular laminin trimers would be deposited in the maturing trophoblastic BM before other isoforms. However, all the 2,
5,
1,
2, and
1 laminin chains were distributed co-extensively at the tip of the mesenchymal core.
In contrast to trophoblastic sprouts, abundant ECM proteins are found among extravillous trophoblastic cells in trophoblastic columns and islands (
Villous Stroma and Capillaries
Distinct changes in the distribution of laminin chains during the development of the villous capillaries were noted. Whereas laminin 2,
1,
2, and
1 chains, possibly as laminin-2 and laminin-4, were found in the capillary BMs throughout placental development,
5 was seen only in the BMs of second- and third-trimester villous capillaries. Both laminin
2 and
5 chains are also found in brain capillary BMs (
2 chain (
2 chain is not found in BMs of villous capillaries. In the latter study, the anti-
2 chain MAb was the same as used in this study. Pepsin-digested, paraffin-embedded tissues were studied, whereas we used acetone-fixed frozen sections; this difference may account for the discrepancy.
EDB and oncofetal fibronectin were found in capillaries in first- but not third-trimester placental villi, possibly reflecting their role in angiogenesis. Interestingly, EDBfibronectin is specifically found in tumors and other pathological processes at sites of neovascularization (
The results of this study show that several laminin and fibronectin isoforms are found in the villous trophoblastic and capillary BMs and in the villous stroma, and that the distribution of some of these ECM molecules is regulated during trophoblastic and capillary BM maturation. During outgrowth of new villous structures, laminins are not found in the trophoblastic sprouts, but they are deposited at the trophoblastmesenchyme interface as the sprouts are invaded by mesenchyme. This suggests that epithelialmesenchymal interaction is a prerequisite for the deposition of the villous BM components.
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Acknowledgments |
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Supported by a project grant from the Medical Faculty, University of Helsinki.
We are indebted to Drs R.E. Burgeson, E. Engvall, S.-I. Hakomori, P. Liesi, V. Pallini, J. Sanes, and L. Zardi for kind gifts of antibodies. The skillful technical assistance of Mr Hannu Kamppinen, Mr Reijo Karppinen, and Ms Marja-Leena Piironen is acknowledged.
Received for publication June 22, 2000; accepted October 10, 2000.
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