JOURNAL
OF STRUCTURAL
BIOLOGY
103, 23-33 (1990)
Organization of Intermediate Filaments and Their Association with Collagen-Containing Phagosomes in Mouse Decidual Cells TELMAM.T.ZORN,S~~RGIOF. Department
of
Histology and Embryology,
DE OLIVEIRA,~ AND PAULOA.ABRAHAMSOHN
Institute of Biomedical Sciences, University of S6o Paulo, Caixn Postal 4365, 05508 Scio Pa&o, Brazil Received December 11, 1989
however, that the elevated expression of desmin was correlated with stromal cell decidualization and that this protein could be considered as a marker of decidualization. Changes in the amount and distribution of extracellular matrix components, such as laminin and fibronectin, also occur during decidual cell transformation (Kisalus et al., 1987). These authors also demonstrated synthesis of collagen type IV by explants of human decidua. The accumulation of collagen around rodent decidual cells (Fainstat, 1963; Lobe1 et al., 1965; Martello and Abrahamsohn, 1986) as well as radioautographic evidence (Oliveira, 1985), indicate that rodent endometrial stromal cells continue producing collagen after their transformation into decidual cells. Zorn et al. (1989) showed that collagen fibrils are phagocytosed by mouse decidual cells and proposed this as one of the mechanisms of endometrial remodeling during the pregnancy of the mouse. The cytoplasm that surrounds the membrane-enveloped collagen fibrils is frequently occupied by a network of filaments of about 10 nm in diameter (Zorn et al., 1989). In the present study we examined the distribution of intermediate filaments in the cytoplasm of mouse antimesometrial decidual cells on the seventh day of pregnancy and especially their association with collagen-containing phagosomes.
We have analyzed the distribution of intermediate filaments (IF) in the cytoplasm of mature decidual cells of mice. IF were scattered throughout the cytoplasm of these cells although there was a preferential accumulation around the nuclei. In many cells a large area of the cytoplasm was occupied by a rich network of IF that extended from the perinuclear region toward the cell surface. Thin bundles of IF crossed the cytoplasm without a preferential orientation. IF were also seen in close association with nuclear pore complexes, gap junctions, mitochondria, and lysosomes. A very developed network of IF surrounded phagosomes that contained collagen fibrils. Longitudinal and cross sections of these phagosomes showed a very close association of IF with the 8 igsoAcademic PI-MS, IIN. phagosome membrane. INTRODUCTION
During embryo implantation the mouse endometrium undergoes remarkable modifications as a consequence of the transformation of stromal fibroblast-like cells into decidual cells. These modifications result in the development of a new structure of epithelioid features, the decidua (Finn, 1971; Weitlauf, 1988; Abrahamsohn, 1983,1989). The fully decidualized stromal cells that form the major part of the mouse antimesometrial decidua on the seventh day of pregnancy are polygonal, multinucleated, closely apposed, and joined by intercellular junctions. In rodents the amount of intermediate filaments (IF) increases during the transformation of spindleshaped cells into decidual cells (Tachi et al., 1972; O’Shea et al., 1983; Welsh and Enders, 1985). Vimentin and desmin have been demonstrated in the endometrium of humans and rats (Glasser and Julian, 1986; Glasser et al., 1987; Kisalus et al., 1987). Glasser and Julian (1986) demonstrated, 1 Present address: Department of Cell Biology, National versity of Brasilia, Brasilia, Brazil.
MATERIALSANDMETHODS Female virgin Swiss mice were mated with males of the same strain, and observed each morning for the presence of vaginal plugs. The day on which a vaginal plug was found was designated the first day of pregnancy. Four animals on the seventh day of pregnancy were used in this study. All animals were anesthetized with chloral hydrate. Their abdominal cavity was opened and the whole length of the uteri was excised and immediately immersed in 2.5% glutaraldehyde in 0.125 M sodium cacodylate buffer at pH 7.4, containing 0.1% tannic acid. Part of the specimens were fixed in the same fixative without tannic acid. Implantation and in&implantation sites were cut into small transverse slices and fixed by immersion in
Uni23
1047-8477/90
All
$3.00
Copyright B 1990 by Academic F’rem, Inc. rights of reproduction in any form resarved.
24
ZORN, DE OLIVEIRA,
the same flxative for 24 hr at 4°C. The tissues were then refixed during 2 hr in osmium tetroxide in the same buffer. The tissues were dehydrated in a series of ethanol and embedded in Epon. Ultrathin sections of the antimesometrial decidua were stained with many1 acetate and lead citrate and observed in a JEOL 100 CX II electron microscope. RESULTS
Most of the stroma of the antimesometrial region of the uterus on the seventh day of pregnancy is occupied by fully decidualized cells (mature decidual cells). These cells are polyhedrical with round large nuclei and conspicuous nucleoli. Their cytoplasm is rich in organelles such as granular endoplasmic reticulum, Golgi stacks, mitochondria, polyribosomes, and lysosome-like bodies. Many collagen-containing phagosomes are seen in the cytoplasm of these cells. Intercellular junctions such as gap and adherenstype junctions connect mature decidual cells. The narrow intercellular spaces are occupied by collagen
AND AEUUHAMSOHN
fibrils and ground substance. The above-described cells surround the embryo whereas the remaining stroma is occupied by cells with long processes and separated by wider extracellular spaces (predecidual cells) and, close to the myometrium, by a population of spindle-shaped cells. A remarkable feature of the mature decidual cells is the large amount of cytoskeletal elements such as microtubules (MT), microfilaments (MF), and especially intermediate filaments. In many cells, but not in all, a well-developed network of IF occupies large areas of the cytoplasm. Although the network of IF is scattered throughout the cytoplasm, there is a preferential accumulation around the nucleus (Figs. 1 and 2). These IF, however, do not surround the entire perimeter of the nucleus but often accumulate around one of its hemispheres. The IF are organized in different patterns. Small, well-defined arrrays of IF are frequently arranged
FIGS. 1-14. Electron micrographs of mouse decidual cells on the 7th day of pregnancy. FIG. 1. Intermediate filaments are observed in a perinuclear situation as bundles (arrows) or as a network concentrated hemisphere of the nucleus (*). x 6820.
around one
INTERMEDIATE
FILAMENTS
IN DECIDUAL
CELLS
25
FIG. 2. Higher magnification of the cell seen in Fig. 1. IF form a network in the perinuclear region (*). Bundles of parallel IF’ are close to the nuclear envelope (arrows) and cell surface (arrowhead). Only polyribosomes and small vesicles are present inside the network. The outlined area shows association between a bundle of IF and a mitochondria. x 17 610. FIG. 3. Tangential section of the nuclear surface. Bundles of IF and some MT (*) appear to be in close proximity with the nuclear surface. Note MT in association with the nuclear pore (arrowheads). The arrows indicate frontal sections of nuclear pores. x 33 480.
parallel to the nuclear envelope in the region immediately adjacent to the nuclear surface (Fig. 3). In cross sections of the nuclear envelope, filaments of 9 nm diameter are observed to connect these arrays of
IF to Other clear ation
components of the arrays of IF appear surface in an oblique the IF appear to end
nuclear surface (Fig. 4). to radiate from the nuorientation. In this situvery close to nuclear pore
26
ZOFtN, DE OLIVEIRA,
AND AHSAHAMSOHN
FIG. 4. Perinuclear region showing a bundle of parallel IF (*I. Small filaments (about 9 run diameter) connect an intermedi late filamel nt to electron dense structures of the outer nuclear membrane ku-rows). x 88 820. FIG. 5. A bundle of IF (*) appears to verge upon a nuclear pore complex (arrowhead). Many microtubules are present in the perinucl .ear region. ‘Ihe inset shows the extremity of a microtubule (arrow) in close association with a nuclear pore. x 67 240; inset, x 46 336.
INTERMEDIATE
FILAMENTS
complexes (Fig. 5). More often, however, the IF do not have a preferential direction and form dense meshworks that can extend toward the cell surface, sometimes occupying most of the cytoplasm between the nucleus and the cell surface (Fig. 6). Less frequently, bundles of parallel IF cross the cytoplasm without an apparent connection with the abovedescribed groups. The dense meshworks of IF generally exclude organelles. Polyribosomes and vesicles (coated and uncoated) are the only structures present amid the filaments (Figs. 1 and 6). Microtubules are often associated to IF situated in the perinuclear cytoplasm. The MT cross the arrays of IF that are parallel to the nuclear surface or they have the same direction of the IF, when these are oblique to the nuclear surface. Similar to the IF, the MT also end near nuclear pore complexes (Fig. 5). A minority of MT are parallel to the nuclear envelope and have unusual sinuous courses (Fig. 7). Small filamentous structures measuring about 9 nm in diameter are anchored to these MT and appear to connect them to other cytoplasmic components (Fig. 7, inset). Microfilaments are also found under the surface of decidual cells, usually forming bundles that are parallel to the plasmalemma. Remarkable concentrations of IF are associated with collagen-containing phagosomes. These phagosomes are situated in different places of the cytoplasm but more frequently in the perinuclear region (Fig. 8). Longitudinal and cross sections of the membranebounded collagen fibrils show the arrangement of the IF in relation to phagosomes. Some IF are parallel to the fibrils whereas most IF are perpendicular or oblique to the collagen fibrils. As a result of this arrangement most phagosomes appear embedded within the IF network, whose filaments interlace with the phagosomes (Fig. 8). The ends of IF are closely associated with the phagosome membranes (Figs. 8 and 9). S ma 11vesicles surround the collagencontaining phagosomes and, surprisingly, some of them seem to be inside the phagosomes. The latter images resemble a multivesicular body as can be seen in Figs. 10 and 11. A few MT are seen around the phagosomes. Intermediate filaments are always found in areas of the cell surface where the plasmalemma forms gap junctions (Fig. 12). IF are also associated to ves-
IN DECIDUAL
27
CELLS
icles of Golgi stacks as well as to mitochondria and lysosome-like dense bodies (Figs. 2, 13, and 14). DISCUSSION
Although IF are considered to be a characteristic feature of rodent decidual cells (Tachi et al., 1972; O’Shea et al., 1983; Glasser and Julian, 1986) details of their distribution and organization have not been reported. Our results show that the perinuclear cytoplasm is the commonest location of a network of IF. It is known that cells of mesenchymal origin, as is the case of the decidual cells, contain abundant IF. Vimentin is the IF protein present in most of these cells, including decidual cells (Steiner-t et al., 1984; Glasser and Julian, 1986; Kisalus et al., 1987). It has been recently shown that different types of IF proteins could be expressed in the same cell (Quinlan and Franke, 1982; Ngai et al., 1986). Glasser et al. (1987) demonstrated that in addition to vimentin, rat decidual cells synthesize a great amount of desmin. This latter protein has been proposed as a marker of decidual cells (Glasser and Julian, 1986; Glasser et al., 1987). Intermediate filaments are thought to be involved in a series of important cellular functions. One of the most important roles of IF is the maintenance of the cell shape (Zackroff et al., 1982). This is demonstrated, for instance, by the dramatic modifications in the arrangement of IF after trypsinization of cells grown in vitro (Goldman et al., 1986). Endometrial stromal fibroblasts undergo a remarkable change in shape while they transform into polygonal decidual cells that become closely apposed by means of intercellular junctions and arranged as an epithelioid structure. Other functions in which IF are probably involved are the movement of organelles, nuclear centration, and cell-cell contact (Steiner% et al., 1984). The preferential localization of IF in the perinuclear region of mouse decidual cells suggests that IF play a role in the centration of their nuclei as has been assumed to occur in other cell types (Goldman and Follet, 1970; Jones et al., 1982). This may be more important in the mouse antimesometrial decidua whose cells are mostly multinucleated. The association of IF with nuclear pore complexes present in mouse decidual cells has also been de-
FIG. 6. A well-developed network of IF (*) occupies a large area of the cytoplasm, between the nucleus and the cell surface. N, nucleus; G, gap junction; A, adherens junction. x 25 990. FIG. 7. Microtubules (Mt) parallel to the nuclear surface in the perinuclear region of a decidual cell. Two of them have sinuous courses. The asterisk indicates an accumulation of electron-dense fibrilar material. N, nucleus. The inset shows thin filaments (arrows) connecting a microtubule with cytoplasmic structures, some of them resembling polyribosomes. x 89 2’70; inset, x 89 270. FIG. 8. Collagen-containing phagosome embedded in a network of IF in the perinuclear region (*). Vesicles with different contents are present in the network. Inset: cross section of membrane-enveloped collagen fibrils surrounded by IF (arrows). A long microtubule (arrowhead) is close to the phagosome. N, nucleus. x 33 450; inset, x 45 200.
28
ZOFW, DE OLIVEIRA,
AND AEiRAHAMSOHN
INTERMEDIATE
FILAMENTS
IN
DECIDUAL
CELLS
30
ZOFW, DE OLIVEIFU,
AND ABRAHAMSOHN
FIG. 9. Longitudinally sectioned collagen-containing phagosome (C) inside a network of IF. Many IF that are perpendicular oblique to the phagosome seem to end at the phagosome membrane (arrows). x 99 200.
scribed in cells grown in vitro and this has been thought to indicate the participation of IF in directing the transport of information from the nucleus to the cytoplasm (Fulton et al., 1980; Bonneau et al., 1985). The examination of the sections showed decidual cells with large collections of IF in their cytoplasm, together with cells that had few IF. Welsh and Enders (1985) suggested that in rat decidual cells these differences could result from the plane of section. As in our material the IF were commonly concentrated around one hemisphere of the nuclei, cells with few IF could have been sectioned through the opposite hemisphere. Serial sections of decidual cells should demonstrate the actual distribution of the IF. Other explanations can be brought forth to explain the differences in amount of IF, such as the existence of two subpopulations of decidual cells, one having compact networks of IF and the other containing
or slightly
fewer IF scattered in the cytoplasm and arranged in thin bundles. On the other hand, the accumulation of IF in the cytoplasm has been correlated with cellular regression or degeneration (see references in Ghadially, 1982). The rodent antimesometrial decidua is a very dynamic structure. The first decidual cells appear on Day 5 of pregnancy of the mouse, and the antimesometrial decidua reaches its maximum development on Days 8 and 9. Thereafter this part of the decidua becomes thinner up to the 11th day, when most of it disappears (Katz and Abrahamsohn, 1987). Involution of decidual cells, however, begins already on the 6th or 7th day of pregnancy as cells located close to the embryo slowly degenerate, probably to yield space for the growing embryo. Assuming that some decidual cells actually have much more IF than others, it can be speculated that those cells containing accumulations of IF are beginning to involute.
INTERMEDIATE
FILAMENTS
IN DECIDUAL
CELLS
31
FIGS. 10 and 11. Longitudinal section of a collagen-containing phagosome. Figures 10 and 11 show the extremities of the same phagosome. It is surrounded by IF, most of them parallel to the long axis of the phagosome. Many vesicles are situated inside the phagosome as in a multivesicular body (arrows). x 73 660.
One of the most interesting findings on the mouse decidua was the close association between IF and collagen-containing phagosomes. It is reasonable to suppose that the entry of a collagen fibril (or a bundle of three or four fibrils) into the cytoplasm could jeopardize the structural stability of the cell. A way for the cell to counterbalance this could be the organization of a network of IF around phagocytosed fibrils. We do not know, however, if the networks are built around the phagosomes or whether the cell introduces the fibrils in areas of the cytoplasm where those networks already exist. There is, on the other hand, evidence for a role of IF in the sequestration of materials to be digested within the cell (Earl et al., 1987; Doherty et al., 1987). These authors showed
that internalized membrane and microinjected glycolytic enzymes could be sequestered in a perinuclear site in association with IF, before their degradation by lysosomal enzymes. It is possible that, in decidual cells, the association of IF with phagosomes also precedes the degradation of collagen by lysosoma1 enzymes. Phagocytosis of collagen has been observed in spindle-shaped cells of the endometrial stroma of mice on the second day of pregnancy (Zorn et al., 1986). No IF were seen associated to these phagosomes although decidual cells originate from these spindle-shaped cells. Therefore the association of IF with phagosomes is not a general phenomenon. In another well-studied system in which active
32
ZOFtN, DE OLIVEIRA,
AND ABRAHAMSOHN
INTERMEDIATE
FILAMENTS
phagocytosis of collagen occurs (gingival fibroblasts), microfilaments were the only cytoskeletal component found associated with phagosomes (Melcher and Chan, 1981). In our material we could not observe a remarkable association of MF with what appeared to be totally internalized collagen fibrils. It could be possible that MF participate actively during the initial stages of engulfment of collagen, as shown by Melcher and Chan (1981), but not in later stages. The microtubules found in the network of IF may be important for the transport of small vesicles that always surround the phagosomes (see also Melcher and Chan, 1981). These vesicles may contain lysosomal enzymes as it has been shown by cytochemistry that collagen-containing phagosomes of mouse decidual cells exhibit acid phosphatase activity (Zorn et al., 1989). We kindly thank Ana Lucia Mota, tiao C. Silva for technical assistance typing the manuscript. This work grants from FAPESP and FINEP.
Gaspar F. Lima, and Sebasand Eliana A. Arduino for was done with the help of
REFERENCES AE~RAHAMSOHN,
P. A. (1983)
Anat.
Embryol.
166, 263-274.
ABRAHAMSOHN, P. A. (1989) in YOSHINAGA, K. (Ed.), Blastocyst Implantation, Morphology of the Decidua, pp. 127-133, Adams, Boston. BONNEAU, A. M., DARVEAU, A., AND SONENBERG, N. (1985) J. Cell Biol. 100, 12091218. DOHERTY, F. J., WASSEL, J. A., AND MAYER, R. J. (1987) Biothem. J. 241,793-800. EARL, R. T., MANGIAPANE, E. H., BILLEW, E. E., R. J. (1987) B&hem. J. 241, 809-815. FAINSTAT, T. (1963) Amer. J. Anat. 112, 337370.
AND
MAYER,
FINN, C. A. (1971) Adv. Reprod. Physiol. 5, l-26. FULTON, A. B., WAN, K. M., AND PENMAN, S. (1980) Cell 20,849 857. GHADIALLY, F. N. (1982) Ultrastructural Pathology of the Cell and Matrix, Intracytoplasmic Filaments, pp. 629-686, Butterworths, London. GLASSER, S. R., AND JULIAN, J. (1986) Biol. Reprod. 35, 463-476.
IN
DECIDUAL
33
CELLS
GLASSER, S. R., LAMPELO, S., MUNIR, Different&ion 35, 132-142.
M. I., AND JULIAN,
J. (1987)
GOLDMAN, R. D., AND FOLLET, E. A. C. (1970) Science 169, 286 288. GOLDMAN, R. D., GOLDMAN, A. E., GREEN, K. J., JONES, J. C., JONES, S. M., AND YANG, H-Y. (1986) J. Cell Sci. Suppl. 5, 69-79. JONES, J. C. R., GOLDMAN, A. E., STEINERT, P. M., YUSPA, S. H., AND GOLDMAN, R. D. (1982) Cell Motil. 2, 197-213. KATZ, S., AND ABRAHAMSOHN, P. A. (1987) Anat. Embryol. 176, 251-258. KISALUS, L. L., HERR, J. C., AND LISLE, 218, 402-415. LOBEL, B. L., TIC, L., AND SHELESNYAK, docrinol. 50, 469-485.
C. D. (1987)
MARTELLO, E. M. V. G., ABRAHAMSOHN, 127, 146-150.
P. A. (1986)
M. C. (1965)
Anat. Actu. Acta
Rec. EnA&.
MELCHER, A. H., AND CHAN, J. (1981) J. Utrustruct. Res. 77, l-36. NGAI, J., CAPETANAKI, Y. G., AND LAZARIDES, E. (1986) Ann. N.Y. Acad. Sci. 455, 144-157. O’SHEA, J. D., KLEINFELD, R. G., AND MORROW, H. A. (1983) Amer. J. Anut. 166, 271-298. OLIVEIRA, S. F. (1985) Incorporaclo de Prolina-H3 pelas celulas da decidua antimesometrial de camundonga durante o quinto e sexto dias de gestacao. Estudo radioautografico. Dissertacao de Mestrado, Instituto de Ciencias Biomedicas, USP, Brasil. QUINLAN, R. A., AND FRANKE, W. W. (1982) Proc. Natl. Acad. Sci. USA 79, 3452-3456. STEINERT, P. M., JONES, J. C. R., AND GOLDMAN, R. D. (1984) J. Cell Biol. 99(1, Pt. 2.), 22s-27s. TACHI, S., TACHI, C., AND LINDNER, H. R. (1972) J. Reprod. Fertil. 31, 59-76. WEITLAUF, H. M. (1988) in. KNOBIL, E., NEILL, J. D., EWING, L. L., MARKERT, C. L., GREENWALD, G. S., AND PFAF, D. W. (Eds.), Physiology of Reproduction: Biology of Implantation, Raven Press, New York. WELSH, A. D., AND ENDERS, A. C. (1985) Amer. 6. Anut. 172, l29. ZACKROFF, R. V., IDLER, W. W., STEINERT, P. M., AND GOLDMAN, R. D. (1982) Proc. Natl. Acad. Sci. USA 79, 754-757. ZORN, T. M. T., BEVILACQUA, E. M. A. F., AND ABRAHAMSOHN, P. A. (1986) Cell Tissue Res. 244, 443-448. ZORN, T. M. T., BJJOVSKY, A. T., BEVILACQUA, E. M. A. F., AND ABRAHAMSOHN, P. A. (1989) A&. Rec. 225, 96100.
FIGS. 12-14. Association between IF and cellular structures. FIG. 12. Vesicles (arrow) close to Golgi stacks (G) are associated with a small bundle FIG. 13. IF (*) appear associated with a lysosome-like dense body. x 68 200. FIG. 14. Bundles of IF (arrow) are seen to be connected with an annular gap junction.
of parallel x 35 220.
IF (*). N, nucleus.
x 49 600.
OF STRUCTURAL
BIOLOGY
103, 23-33 (1990)
Organization of Intermediate Filaments and Their Association with Collagen-Containing Phagosomes in Mouse Decidual Cells TELMAM.T.ZORN,S~~RGIOF. Department
of
Histology and Embryology,
DE OLIVEIRA,~ AND PAULOA.ABRAHAMSOHN
Institute of Biomedical Sciences, University of S6o Paulo, Caixn Postal 4365, 05508 Scio Pa&o, Brazil Received December 11, 1989
however, that the elevated expression of desmin was correlated with stromal cell decidualization and that this protein could be considered as a marker of decidualization. Changes in the amount and distribution of extracellular matrix components, such as laminin and fibronectin, also occur during decidual cell transformation (Kisalus et al., 1987). These authors also demonstrated synthesis of collagen type IV by explants of human decidua. The accumulation of collagen around rodent decidual cells (Fainstat, 1963; Lobe1 et al., 1965; Martello and Abrahamsohn, 1986) as well as radioautographic evidence (Oliveira, 1985), indicate that rodent endometrial stromal cells continue producing collagen after their transformation into decidual cells. Zorn et al. (1989) showed that collagen fibrils are phagocytosed by mouse decidual cells and proposed this as one of the mechanisms of endometrial remodeling during the pregnancy of the mouse. The cytoplasm that surrounds the membrane-enveloped collagen fibrils is frequently occupied by a network of filaments of about 10 nm in diameter (Zorn et al., 1989). In the present study we examined the distribution of intermediate filaments in the cytoplasm of mouse antimesometrial decidual cells on the seventh day of pregnancy and especially their association with collagen-containing phagosomes.
We have analyzed the distribution of intermediate filaments (IF) in the cytoplasm of mature decidual cells of mice. IF were scattered throughout the cytoplasm of these cells although there was a preferential accumulation around the nuclei. In many cells a large area of the cytoplasm was occupied by a rich network of IF that extended from the perinuclear region toward the cell surface. Thin bundles of IF crossed the cytoplasm without a preferential orientation. IF were also seen in close association with nuclear pore complexes, gap junctions, mitochondria, and lysosomes. A very developed network of IF surrounded phagosomes that contained collagen fibrils. Longitudinal and cross sections of these phagosomes showed a very close association of IF with the 8 igsoAcademic PI-MS, IIN. phagosome membrane. INTRODUCTION
During embryo implantation the mouse endometrium undergoes remarkable modifications as a consequence of the transformation of stromal fibroblast-like cells into decidual cells. These modifications result in the development of a new structure of epithelioid features, the decidua (Finn, 1971; Weitlauf, 1988; Abrahamsohn, 1983,1989). The fully decidualized stromal cells that form the major part of the mouse antimesometrial decidua on the seventh day of pregnancy are polygonal, multinucleated, closely apposed, and joined by intercellular junctions. In rodents the amount of intermediate filaments (IF) increases during the transformation of spindleshaped cells into decidual cells (Tachi et al., 1972; O’Shea et al., 1983; Welsh and Enders, 1985). Vimentin and desmin have been demonstrated in the endometrium of humans and rats (Glasser and Julian, 1986; Glasser et al., 1987; Kisalus et al., 1987). Glasser and Julian (1986) demonstrated, 1 Present address: Department of Cell Biology, National versity of Brasilia, Brasilia, Brazil.
MATERIALSANDMETHODS Female virgin Swiss mice were mated with males of the same strain, and observed each morning for the presence of vaginal plugs. The day on which a vaginal plug was found was designated the first day of pregnancy. Four animals on the seventh day of pregnancy were used in this study. All animals were anesthetized with chloral hydrate. Their abdominal cavity was opened and the whole length of the uteri was excised and immediately immersed in 2.5% glutaraldehyde in 0.125 M sodium cacodylate buffer at pH 7.4, containing 0.1% tannic acid. Part of the specimens were fixed in the same fixative without tannic acid. Implantation and in&implantation sites were cut into small transverse slices and fixed by immersion in
Uni23
1047-8477/90
All
$3.00
Copyright B 1990 by Academic F’rem, Inc. rights of reproduction in any form resarved.
24
ZORN, DE OLIVEIRA,
the same flxative for 24 hr at 4°C. The tissues were then refixed during 2 hr in osmium tetroxide in the same buffer. The tissues were dehydrated in a series of ethanol and embedded in Epon. Ultrathin sections of the antimesometrial decidua were stained with many1 acetate and lead citrate and observed in a JEOL 100 CX II electron microscope. RESULTS
Most of the stroma of the antimesometrial region of the uterus on the seventh day of pregnancy is occupied by fully decidualized cells (mature decidual cells). These cells are polyhedrical with round large nuclei and conspicuous nucleoli. Their cytoplasm is rich in organelles such as granular endoplasmic reticulum, Golgi stacks, mitochondria, polyribosomes, and lysosome-like bodies. Many collagen-containing phagosomes are seen in the cytoplasm of these cells. Intercellular junctions such as gap and adherenstype junctions connect mature decidual cells. The narrow intercellular spaces are occupied by collagen
AND AEUUHAMSOHN
fibrils and ground substance. The above-described cells surround the embryo whereas the remaining stroma is occupied by cells with long processes and separated by wider extracellular spaces (predecidual cells) and, close to the myometrium, by a population of spindle-shaped cells. A remarkable feature of the mature decidual cells is the large amount of cytoskeletal elements such as microtubules (MT), microfilaments (MF), and especially intermediate filaments. In many cells, but not in all, a well-developed network of IF occupies large areas of the cytoplasm. Although the network of IF is scattered throughout the cytoplasm, there is a preferential accumulation around the nucleus (Figs. 1 and 2). These IF, however, do not surround the entire perimeter of the nucleus but often accumulate around one of its hemispheres. The IF are organized in different patterns. Small, well-defined arrrays of IF are frequently arranged
FIGS. 1-14. Electron micrographs of mouse decidual cells on the 7th day of pregnancy. FIG. 1. Intermediate filaments are observed in a perinuclear situation as bundles (arrows) or as a network concentrated hemisphere of the nucleus (*). x 6820.
around one
INTERMEDIATE
FILAMENTS
IN DECIDUAL
CELLS
25
FIG. 2. Higher magnification of the cell seen in Fig. 1. IF form a network in the perinuclear region (*). Bundles of parallel IF’ are close to the nuclear envelope (arrows) and cell surface (arrowhead). Only polyribosomes and small vesicles are present inside the network. The outlined area shows association between a bundle of IF and a mitochondria. x 17 610. FIG. 3. Tangential section of the nuclear surface. Bundles of IF and some MT (*) appear to be in close proximity with the nuclear surface. Note MT in association with the nuclear pore (arrowheads). The arrows indicate frontal sections of nuclear pores. x 33 480.
parallel to the nuclear envelope in the region immediately adjacent to the nuclear surface (Fig. 3). In cross sections of the nuclear envelope, filaments of 9 nm diameter are observed to connect these arrays of
IF to Other clear ation
components of the arrays of IF appear surface in an oblique the IF appear to end
nuclear surface (Fig. 4). to radiate from the nuorientation. In this situvery close to nuclear pore
26
ZOFtN, DE OLIVEIRA,
AND AHSAHAMSOHN
FIG. 4. Perinuclear region showing a bundle of parallel IF (*I. Small filaments (about 9 run diameter) connect an intermedi late filamel nt to electron dense structures of the outer nuclear membrane ku-rows). x 88 820. FIG. 5. A bundle of IF (*) appears to verge upon a nuclear pore complex (arrowhead). Many microtubules are present in the perinucl .ear region. ‘Ihe inset shows the extremity of a microtubule (arrow) in close association with a nuclear pore. x 67 240; inset, x 46 336.
INTERMEDIATE
FILAMENTS
complexes (Fig. 5). More often, however, the IF do not have a preferential direction and form dense meshworks that can extend toward the cell surface, sometimes occupying most of the cytoplasm between the nucleus and the cell surface (Fig. 6). Less frequently, bundles of parallel IF cross the cytoplasm without an apparent connection with the abovedescribed groups. The dense meshworks of IF generally exclude organelles. Polyribosomes and vesicles (coated and uncoated) are the only structures present amid the filaments (Figs. 1 and 6). Microtubules are often associated to IF situated in the perinuclear cytoplasm. The MT cross the arrays of IF that are parallel to the nuclear surface or they have the same direction of the IF, when these are oblique to the nuclear surface. Similar to the IF, the MT also end near nuclear pore complexes (Fig. 5). A minority of MT are parallel to the nuclear envelope and have unusual sinuous courses (Fig. 7). Small filamentous structures measuring about 9 nm in diameter are anchored to these MT and appear to connect them to other cytoplasmic components (Fig. 7, inset). Microfilaments are also found under the surface of decidual cells, usually forming bundles that are parallel to the plasmalemma. Remarkable concentrations of IF are associated with collagen-containing phagosomes. These phagosomes are situated in different places of the cytoplasm but more frequently in the perinuclear region (Fig. 8). Longitudinal and cross sections of the membranebounded collagen fibrils show the arrangement of the IF in relation to phagosomes. Some IF are parallel to the fibrils whereas most IF are perpendicular or oblique to the collagen fibrils. As a result of this arrangement most phagosomes appear embedded within the IF network, whose filaments interlace with the phagosomes (Fig. 8). The ends of IF are closely associated with the phagosome membranes (Figs. 8 and 9). S ma 11vesicles surround the collagencontaining phagosomes and, surprisingly, some of them seem to be inside the phagosomes. The latter images resemble a multivesicular body as can be seen in Figs. 10 and 11. A few MT are seen around the phagosomes. Intermediate filaments are always found in areas of the cell surface where the plasmalemma forms gap junctions (Fig. 12). IF are also associated to ves-
IN DECIDUAL
27
CELLS
icles of Golgi stacks as well as to mitochondria and lysosome-like dense bodies (Figs. 2, 13, and 14). DISCUSSION
Although IF are considered to be a characteristic feature of rodent decidual cells (Tachi et al., 1972; O’Shea et al., 1983; Glasser and Julian, 1986) details of their distribution and organization have not been reported. Our results show that the perinuclear cytoplasm is the commonest location of a network of IF. It is known that cells of mesenchymal origin, as is the case of the decidual cells, contain abundant IF. Vimentin is the IF protein present in most of these cells, including decidual cells (Steiner-t et al., 1984; Glasser and Julian, 1986; Kisalus et al., 1987). It has been recently shown that different types of IF proteins could be expressed in the same cell (Quinlan and Franke, 1982; Ngai et al., 1986). Glasser et al. (1987) demonstrated that in addition to vimentin, rat decidual cells synthesize a great amount of desmin. This latter protein has been proposed as a marker of decidual cells (Glasser and Julian, 1986; Glasser et al., 1987). Intermediate filaments are thought to be involved in a series of important cellular functions. One of the most important roles of IF is the maintenance of the cell shape (Zackroff et al., 1982). This is demonstrated, for instance, by the dramatic modifications in the arrangement of IF after trypsinization of cells grown in vitro (Goldman et al., 1986). Endometrial stromal fibroblasts undergo a remarkable change in shape while they transform into polygonal decidual cells that become closely apposed by means of intercellular junctions and arranged as an epithelioid structure. Other functions in which IF are probably involved are the movement of organelles, nuclear centration, and cell-cell contact (Steiner% et al., 1984). The preferential localization of IF in the perinuclear region of mouse decidual cells suggests that IF play a role in the centration of their nuclei as has been assumed to occur in other cell types (Goldman and Follet, 1970; Jones et al., 1982). This may be more important in the mouse antimesometrial decidua whose cells are mostly multinucleated. The association of IF with nuclear pore complexes present in mouse decidual cells has also been de-
FIG. 6. A well-developed network of IF (*) occupies a large area of the cytoplasm, between the nucleus and the cell surface. N, nucleus; G, gap junction; A, adherens junction. x 25 990. FIG. 7. Microtubules (Mt) parallel to the nuclear surface in the perinuclear region of a decidual cell. Two of them have sinuous courses. The asterisk indicates an accumulation of electron-dense fibrilar material. N, nucleus. The inset shows thin filaments (arrows) connecting a microtubule with cytoplasmic structures, some of them resembling polyribosomes. x 89 2’70; inset, x 89 270. FIG. 8. Collagen-containing phagosome embedded in a network of IF in the perinuclear region (*). Vesicles with different contents are present in the network. Inset: cross section of membrane-enveloped collagen fibrils surrounded by IF (arrows). A long microtubule (arrowhead) is close to the phagosome. N, nucleus. x 33 450; inset, x 45 200.
28
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ZOFW, DE OLIVEIFU,
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FIG. 9. Longitudinally sectioned collagen-containing phagosome (C) inside a network of IF. Many IF that are perpendicular oblique to the phagosome seem to end at the phagosome membrane (arrows). x 99 200.
scribed in cells grown in vitro and this has been thought to indicate the participation of IF in directing the transport of information from the nucleus to the cytoplasm (Fulton et al., 1980; Bonneau et al., 1985). The examination of the sections showed decidual cells with large collections of IF in their cytoplasm, together with cells that had few IF. Welsh and Enders (1985) suggested that in rat decidual cells these differences could result from the plane of section. As in our material the IF were commonly concentrated around one hemisphere of the nuclei, cells with few IF could have been sectioned through the opposite hemisphere. Serial sections of decidual cells should demonstrate the actual distribution of the IF. Other explanations can be brought forth to explain the differences in amount of IF, such as the existence of two subpopulations of decidual cells, one having compact networks of IF and the other containing
or slightly
fewer IF scattered in the cytoplasm and arranged in thin bundles. On the other hand, the accumulation of IF in the cytoplasm has been correlated with cellular regression or degeneration (see references in Ghadially, 1982). The rodent antimesometrial decidua is a very dynamic structure. The first decidual cells appear on Day 5 of pregnancy of the mouse, and the antimesometrial decidua reaches its maximum development on Days 8 and 9. Thereafter this part of the decidua becomes thinner up to the 11th day, when most of it disappears (Katz and Abrahamsohn, 1987). Involution of decidual cells, however, begins already on the 6th or 7th day of pregnancy as cells located close to the embryo slowly degenerate, probably to yield space for the growing embryo. Assuming that some decidual cells actually have much more IF than others, it can be speculated that those cells containing accumulations of IF are beginning to involute.
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FIGS. 10 and 11. Longitudinal section of a collagen-containing phagosome. Figures 10 and 11 show the extremities of the same phagosome. It is surrounded by IF, most of them parallel to the long axis of the phagosome. Many vesicles are situated inside the phagosome as in a multivesicular body (arrows). x 73 660.
One of the most interesting findings on the mouse decidua was the close association between IF and collagen-containing phagosomes. It is reasonable to suppose that the entry of a collagen fibril (or a bundle of three or four fibrils) into the cytoplasm could jeopardize the structural stability of the cell. A way for the cell to counterbalance this could be the organization of a network of IF around phagocytosed fibrils. We do not know, however, if the networks are built around the phagosomes or whether the cell introduces the fibrils in areas of the cytoplasm where those networks already exist. There is, on the other hand, evidence for a role of IF in the sequestration of materials to be digested within the cell (Earl et al., 1987; Doherty et al., 1987). These authors showed
that internalized membrane and microinjected glycolytic enzymes could be sequestered in a perinuclear site in association with IF, before their degradation by lysosomal enzymes. It is possible that, in decidual cells, the association of IF with phagosomes also precedes the degradation of collagen by lysosoma1 enzymes. Phagocytosis of collagen has been observed in spindle-shaped cells of the endometrial stroma of mice on the second day of pregnancy (Zorn et al., 1986). No IF were seen associated to these phagosomes although decidual cells originate from these spindle-shaped cells. Therefore the association of IF with phagosomes is not a general phenomenon. In another well-studied system in which active
32
ZOFtN, DE OLIVEIRA,
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phagocytosis of collagen occurs (gingival fibroblasts), microfilaments were the only cytoskeletal component found associated with phagosomes (Melcher and Chan, 1981). In our material we could not observe a remarkable association of MF with what appeared to be totally internalized collagen fibrils. It could be possible that MF participate actively during the initial stages of engulfment of collagen, as shown by Melcher and Chan (1981), but not in later stages. The microtubules found in the network of IF may be important for the transport of small vesicles that always surround the phagosomes (see also Melcher and Chan, 1981). These vesicles may contain lysosomal enzymes as it has been shown by cytochemistry that collagen-containing phagosomes of mouse decidual cells exhibit acid phosphatase activity (Zorn et al., 1989). We kindly thank Ana Lucia Mota, tiao C. Silva for technical assistance typing the manuscript. This work grants from FAPESP and FINEP.
Gaspar F. Lima, and Sebasand Eliana A. Arduino for was done with the help of
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MELCHER, A. H., AND CHAN, J. (1981) J. Utrustruct. Res. 77, l-36. NGAI, J., CAPETANAKI, Y. G., AND LAZARIDES, E. (1986) Ann. N.Y. Acad. Sci. 455, 144-157. O’SHEA, J. D., KLEINFELD, R. G., AND MORROW, H. A. (1983) Amer. J. Anut. 166, 271-298. OLIVEIRA, S. F. (1985) Incorporaclo de Prolina-H3 pelas celulas da decidua antimesometrial de camundonga durante o quinto e sexto dias de gestacao. Estudo radioautografico. Dissertacao de Mestrado, Instituto de Ciencias Biomedicas, USP, Brasil. QUINLAN, R. A., AND FRANKE, W. W. (1982) Proc. Natl. Acad. Sci. USA 79, 3452-3456. STEINERT, P. M., JONES, J. C. R., AND GOLDMAN, R. D. (1984) J. Cell Biol. 99(1, Pt. 2.), 22s-27s. TACHI, S., TACHI, C., AND LINDNER, H. R. (1972) J. Reprod. Fertil. 31, 59-76. WEITLAUF, H. M. (1988) in. KNOBIL, E., NEILL, J. D., EWING, L. L., MARKERT, C. L., GREENWALD, G. S., AND PFAF, D. W. (Eds.), Physiology of Reproduction: Biology of Implantation, Raven Press, New York. WELSH, A. D., AND ENDERS, A. C. (1985) Amer. 6. Anut. 172, l29. ZACKROFF, R. V., IDLER, W. W., STEINERT, P. M., AND GOLDMAN, R. D. (1982) Proc. Natl. Acad. Sci. USA 79, 754-757. ZORN, T. M. T., BEVILACQUA, E. M. A. F., AND ABRAHAMSOHN, P. A. (1986) Cell Tissue Res. 244, 443-448. ZORN, T. M. T., BJJOVSKY, A. T., BEVILACQUA, E. M. A. F., AND ABRAHAMSOHN, P. A. (1989) A&. Rec. 225, 96100.
FIGS. 12-14. Association between IF and cellular structures. FIG. 12. Vesicles (arrow) close to Golgi stacks (G) are associated with a small bundle FIG. 13. IF (*) appear associated with a lysosome-like dense body. x 68 200. FIG. 14. Bundles of IF (arrow) are seen to be connected with an annular gap junction.
of parallel x 35 220.
IF (*). N, nucleus.
x 49 600.
