Trophoblast-decidual cell interactions and establishment of maternal blood circulation in the parietal yolk sac placenta of the rat.код для вставкиСкачать
THE ANATOMICAL RECORD 217:203-219 (1987) Trophoblast-Decidual Cell Interactions and Establishment of Maternal Blood Circulation in the Parietal Yolk Sac Placenta of the Rat ALERICK 0. WELSH AND ALLEN C. ENDERS Department of Human Anatomy, University of California, Dauis, CA 95616 ABSTRACT Implantation sites from rats were studied on days 6, 7, and 8 of pregnancy to determine the sequence of events in the formation of blood spaces in the trophoblast that is part of the parietal wall of the yolk sac placenta and to determine how trophoblast gains access to maternal blood. The maternal blood flowing through these spaces is the source of nutrients that reach the embryo via the visceral endoderm. Tissues were prepared for light microscopy, scanning electron microscopy, and transmission electron microscopy. Trophoblast blood spaces are derived from the lateral intercellular spaces of trophoblast cells and are present in a collapsed condition until day 8, when maternal vessels are tapped by trophoblast. These spaces then contain circulating maternal blood, and trophoblast cells reflect adaptations for metabolic exchange including thinning of trophoblast covering Reichert’s membrane and the appearance of numerous fenestrations, with and without diaphragms, in the areas where trophoblast is attenuated. Between days 6 and 7 decidual cells appear to form a barrier between the maternal circulation and trophoblast. On day 7, however, decidual cell processes penetrate the residual uterine luminal epithelia1 basal lamina, and then the decidual cells that are juxtaposed to trophoblast undergo degradative changes that resemble apoptosis. There is condensation of cytoplasmic contents, fragmentation of the cells, and phagocytosis of the fragments by trophoblast. Some decidual cells are interposed between endothelial cells in the walls of maternal vessels as early as day 7. Trophoblast may gain access to the maternal vessels by replacing decidual cells or by direct imposition of trophoblast cell processes between endothelial cells. The importance of the visceral yolk sac as a functional placenta in laboratory rodents is well established (Anderson, 1959; Lambson, 1966; Padykula et al., 1966; Jollie, 1968, 1986; Carpenter, 1980). During early gestation, prior to the formation of the complementary chorioallantoic placenta, the yolk sac placenta is especially important since it is the only placental structure present. There have also been some studies about the role that the parietal wall of the yolk sac plays in presenting materials to the visceral wall of the yolk sac via the yolk sac cavity (Everett, 1935; Seibel, 1974; Carpenter, 1980; Parr and Parr, 1986). It has been shown that trophoblast cells form a network of channels that distributes maternal blood throughout the parietal wall of the yolk sac placenta (Everett, 1935).These trophoblast blood spaces are present during midpregnancy (day 8 to day 16 in the rat) and are in the layer of trophoblast cells composing the outermost layer of the parietal wall of the yolk sac (i.e., bilaminar omphalopleure). This layer of trophoblast cells is situated between decidual cells and Reichert’s membrane. Reichert’s membrane probably plays a significant role as a selective barrier to the movement of material from maternal blood (flowing through trophoblast blood spaces) to the yolk sac cavity (Jollie, 1968; Carpenter, 1980). Most studies of parietal yolk sac have been concerned with middle to late gestation periods, and early development of the parietal wall of the yolk 0 1987 ALAN R. LISS, INC sac has not been adequately studied. For example, while it is generally known that the parietal wall of the yolk sac ruptures during the later stages of pregnancy (Bridgman, 1948; Jensh et al., 1977; Dickson, 1979), there is little information in the literature about the formation of trophoblast blood spaces or even the cellular events involved in trophoblast cell migration to the maternal blood supply. In order to tap maternal vessels, trophoblast cells must penetrate or displace the uterine luminal epithelium and the layers of decidual cells separating it from the maternal vessels. There has been some controversy about the degree of invasiveness of trophoblast cells through both the uterine luminal epithelium and the decidualized uterine stroma. Many studies have favored the concept that penetration of the luminal epithelium in rats and mice is a result of programmed cell death (Finn and Hinchliffe, 1964; Enders and Schlafke, 1967; Finn and Bredl, 1973; El-Shershaby and Hinchliffe, 1974, 1975) and not a result of cytolytic invasion of trophoblast. It is commonly agreed, however, that trophoblast is phagocytic (Sherman and Wudl, 1976). El-Shershaby and Hinchliffe (1975) proposed that uterine luminal penetration in the mouse involves autolysis or self-digestion accompanied by trophoblast phagocytosis. In their ultraReceived June 19,1986; accepted October 6, 1986. 204 A.O. WELSH AND A.C. ENDERS structural study they found little evidence of lysosomal activity in trophoblast cells prior to uterine epithelial deterioration but did find evidence of autolysosomes in the uterine epithelial cells. Although a few studies have focused on the presence of lysosomes and lysosomal enzymes in trophoblast and uterine luminal epithelium (Christie, 1967; Hall, 1971; Elangovan and Moulton, 1980), to date there have been no ultrastructural cytochemical studies performed in order to localize lysosomal enzymes during normal implantation. Unfortunately the degeneration of the uterine luminal epithelium, its penetration by trophoblast, and the later stromal cell development have not been studied in light of more recent concepts of cell death (Wyllie, 1981). The lytic potential of trophoblast has also been brought into question with regard to penetration of the uterine luminal basal lamina. That trophoblast penetration of the uterine stroma of rats and mice is delayed somewhat a t the uterine luminal basal lamina has been known for some time (Enders and Schlafke, 1967; Potts, 1969) and is generally accepted (Glasser, 1985). Only recently, however, has it been shown that in the rat, decidual cell processes rather than trophoblast cell processes penetrate the basal lamina (Schlafke et al., 1985).Here again the active agents in the disruption of a potential barrier to trophoblast migration are not well understood but imply a n active role by maternal tissues. Once decidual cells have penetrated the basal lamina they come into direct contact with trophoblast cells. Although many light microscopic studies have demonstrated the invasive characteristics of rodent trophoblast in a variety of ectopic sites (see review by Sherman and Wudl, 19761, there have been few morphological studies dealing with the interactions of trophoblast and normal decidua. Those that do consider this subject do so indirectly and derive their information from paraffinembedded material (Kirby and Cowell, 1968; Billington, 1971). To clarify some of the events of trophoblast blood space formation, trophoblast penetration of decidua, and tapping of maternal vessels, we have studied rat implantation sites on days 6, 7, and 8 of normal pregnancy. Previous studies from this laboratory have examined trophoblast cell-epithelia1 cell interactions (Enders and Schlafke, 1967), basal lamina penetration (Schlafke et al., 1985), and the structure of mature decidua (Welsh and Enders, 1983). MATERIALS AND METHODS Primigravid Sprague-Dawley rats used for this study were maintained on a light cycle of 14-hour light and 10hour dark. Males were placed overnight in cages with two females, and females with positive vaginal smears the following morning were considered to be in day 1of pregnancy. Implantation sites were collected on days 6, Fig. 2. By day 7 many changes have occurred at the interface of maternal and embryonic tissues. Trophoblast cells (Tr)overlap one another extensively with very little extracellular space separating them (between arrows in 2 and 2a). In Figure 2a, a higher magnification of a region similar to that shown in Figure 2, fenestrations that first appear on day 7 are seen (arrowheads) in the thin trophoblast flanges. The decidual cells (Dec) are elongated along the mesometrialantimesometrial axis in Figure 2 and a decidual cell process (Pr)is penetrating the uterine luminal basal lamina (BL) in Figure 2a. Note also the presence of collagen (C) in the spaces between decidual cell processes and between the decidual cell process and the trophoblast cell. Longitudinal section. Day 7, 2 P.M. Figure 2, ~ 9 , 0 7 0Figure . 2a, x 16,600. Fig. 1 . This electron micrograph illustrates some of the earliest features of trophoblast blood space formation. Processes on the basal-lateral surfaces of adjacent trophoblast cells (Tr) interdigitate (upper right) and the lateral intercellular space is somewhat open. Just below the lateral intercellular space there is an apical junctional complex connecting the trophoblast cells (arrow). At this time trophoblast cells are in close association with the apical surfaces of uterine luminal epithelial cells (Ep). Longitudinal section. Day 6 , 2 P.M. x 11,180. TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT 205 206 A.O. WELSH AND A.C. ENDERS Fig. 3. This is a higher magnification of a region similar to that in the previous micrograph. The trophoblast cells (Tr) are separated by a small extracellular space (arrows) and the thin flange of the trophoblast cell adjacent to Reichert’s membrane (RM) is fenestrated (arrowheads). In the center portion of the micrograph a process (Pr)of a decidual cell (Dec) is penetrating the residual uterine lumina1 basal lamina (BL). There is flocculent material of moderate electron density in the extracellular space between folds and processes of decidual cells. Longitudinal section. Day 7 , 2 P.M. ~20,800. 7, and 8. Although most tissues were collected a t 2 P.M., some were collected a t 2 A.M., 8 A.M., and 8 P.M. Animals were anesthetized with ether, laparotomized, and perfused with Dulbecco’s phosphate-buffered saline (Gibco, Grand Island, NY) followed by fixative with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). Perfusion was performed as previously described (Welsh and Enders, 1985). Isolated implantation sites were fixed for a total of 1.5-2 hours. Some sites were carefully trimmed and postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer, pH 7.3, for 1-2 hours. These were dehydrated in ethanol and propylene oxide and embedded in Durcupan Araldite resin. Thick sections (1-2 pm) were stained with Azure B, and thin sections cut on a Sorvall MT-2 or MT-5000 ultramicrotome were stained with uranyl acetate and lead citrate. Electron micrograph negatives were taken with a Zeiss EM-10 or a Philips 410 electron microscope. 207 TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT variably open at the basal-lateral border (Fig. 1).The basal surface of trophoblast abuts a basal lamina that is destined to become part of Reichert’s membrane. Basallateral processes from adjacent trophoblast cells interdigitate with one another in the basal-lateral compartment and also frequently overlap one another. With further development the basal-lateral juxtaposition of cells becomes more frequent and the overlapping more extensive. Day 7 Fig. 4. Extensive development of decidual cells (arrows)in the region adjacent to the conceptus appears to promote the separation of maternal blood vessels from the conceptus. Longitudinal section. Day 7, 2 P.M. X130. Other sites were cut with a razor blade and torn in a manner that would expose the conceptus, extraembryonic membranes, and uterine stroma. These were also postfixed in 2% osmium tetroxide in phosphate buffer, dehydrated in ethanol, placed in acetone, and critical-point dried with COZ. Specimens were selected under a dissecting microscope, mounted on aluminum stubs, and coated with gold in a metal evaporator, and photomicrographs were taken with a Philips SEM 501 scanning electron microscope. OBSERVATIONS Since the first trophoblast blood spaces are derived from the lateral intercellular spaces of mural trophoblast, their formation begins with the formation of mural trophoblast of the blastocyst. Early in gestation (days 5 and 6), these lateral spaces are demarcated by junctional complexes on the apical-lateral border and are At 2 P.M. on day 7 thin flanges from trophoblast cells may extend for great distances across the apical-lateral and basal-lateral surfaces of adjacent cells (Fig. 2). In other areas the shorter flanges or processes mentioned above persist. There is usually little intercellular space between adjacent trophoblast cells. By 2 P.M. on day 7 fenestrations appear in trophoblast cell flanges (Figs. 2, 3). These tend to develop earlier and more extensively in the flanges on the basal surfaces apposed to Reichert’s membrane. The fenestrations vary in diameter (60-90 nm) and usually appear to have a diaphragm. In places wider gaps are present and no diaphragm is observed. Shorter flanges that project into the lateral and basal compartments also have fenestrations. By the afternoon of day 7 the luminal epithelium in the lateral walls of the implantation chamber is gone. There are still epithelial cells in the mesometrial, antimesometrial, proximal, and distal areas of the implantation chamber. The antimesometrial decidual cells are well developed and appear to have segregated the maternal vasculature from the trophoblast by some distance (Fig. 4). Decidual cells in the region next to the implantation chamber are tightly packed with extensive areas of membrane apposition between cells (Fig. 2). Although it appears that membrane specializations may occur (Figs. 5, 7a), no attempt was made to characterize them in this study. Collagen bundles occur often between apposed decidual cells (Fig. 5). There are lysosomelike granules and granules with a heterogenous matrix (Figs. 3, 5). Golgi elements, vesicles, and other small granules are numerous. Nuclei are somewhat irregular in outline and contain a moderate amount of heterochromatin and prominent nucleoli. On the afternoon of day 7 the decidua appears healthy with few signs of cellular degeneration except in the decidual crypt (Welsh and Enders, 1983). At this time the layer of decidual cells lining the implantation chamber is polarized. Polar views of the implantation chamber (i.e., sections cut perpendicular to the mesometrial-antimesometrial axis of the implantation chamber) demonstrate that the apical surface is thrown into folds and processes that surround amorphous extracellular matrix or bundles of collagen or both (Fig. 5). The collagen bundles are oriented along a mesometrial-antimesometrial axis. Some processes extend through the residual uterine luminal basal lamina and contact trophoblast cells. Following this penetration it is common to find collagen fibrils on the luminal side of the uterine luminal epithelial basal lamina between decidual cell processes and trophoblast and between decidual cell processes and the basal lamina (Fig. 2a). There are numerous intermediate filaments and microtubules in these decidual cells, and they are generally oriented along the mesometrial-antimesometrial axis. A.O. WELSH AND A.C. ENDERS 208 Fig. 5. When polar sections of the implantation chamber are studied, a high degree of cellular and extracellular organization becomes evident. In this micrograph, fields of microtubules (MT)and intermediate filaments (F) are seen in cross section in the decidual cells. Processes or flanges at decidual cell surfaces facing trophoblast are wrapped around collagen fibrils that are oriented in a mesometrial-antimeso- metrial direction. These processes occur primarily at one surface and give the decidual cells polarity. There is a decidual cell process on the luminal side of the uterine luminal basal lamina (arrows) indicating penetration of the basal lamina. Note the increased density in the regions where trophoblast and decidual cell membranes are closely apposed. Polar section. Day 7,2 P.M. ~28,700. These cells often contain lipid droplets and areas of concentrated glycogen. The mitochondria are of intermediate electron density with lamellar cristae. There are numerous stacks of rough endoplasmic reticula @ER). contain a few maternal blood cells and platelets but are mostly open spaces (Figs. 6,7,10). In nonperfused specimens the spaces are packed with maternal blood cells or collapsed. Trophoblast cell bodies may be juxtaposed to decidual cells or Reichert’s membrane or individual cells may span the blood space and be juxtaposed to both. Trophoblast cell processes cover the areas of Reichert’s membrane and regions of decidual cells not in contact with trophoblast cell bodies (Fig. 7). Moreover, these processes tend to cover Reichert’s membrane more completely than the decidual cell surface. Processes are usually thin and fenestrated, and many of the larger fenestrations do not have diaphragms. Frequently decidual cells form part of the wall of trophoblast blood spaces (Fig. 7a), although their contribution is never as great a s that of trophoblast cells. Fenestrated trophoblast Day 8 Prior to day 8 the plasma membranes of adjacent trophoblast cells are variably close together, and trophoblast spaces are only potential blood spaces. By the afternoon of day 8, however, after trophoblast cells tap the maternal vessels and blood begins flowing through these spaces, they open up to become large channels that are limited by the lateral cell membranes of trophoblast cells and trophoblast cell processes. After vascular perfusion the spaces within the trophoblast layer may Fig. 6. This light micrograph was prepared from a day 8 implantation site. Note that the embryo occupies only the central portion of the implantation chamber. The upper box indicates the region shown at a higher magnification in Figure 6a and the lower box indicates Figure 6b. Figure 6a illustrates the opening of a maternal blood vessel into the former uterine lumen (arrow). Most of the uterine lumina1 epithelium has been sloughed into the lumen and is degenerating. Figure 6b shows the presence of trophoblast blood spaces VBS) that have been cleared of maternal blood during perfusion fixation. The decidual cells (Dec) in the regions next to the trophoblast (Tr) are more flattened, darker, and smaller, indicating the onset of apoptosis. The parietal endoderm (PE) and visceral endoderm N E ) are evident. Longitudinal section. Day 8 , 2 P.M. Figure 6, ~ 8 0Figure . 6a, ~ 3 0 0Figure . 6b, ~ 3 0 0 . 210 A.O. WELSH AND A.C. ENDERS Fig. 7. This electron micrograph taken from a region similar to that seen in Figure 6b shows the relationship of decidual cells (Dec),trophoblast (Tr), and parietal endoderm (PE) around a perfused trophoblast blood space (TBS). Trophoblast processes protrude into the trophoblast blood space and are thin and fenestrated. Trophoblast processes that cover Reichert’s membrane are usually thin and fenestrated as well but there are no substantial breaks in the continuity of the trophoblast. The bracket indicates the region shown in Figure 7a where the trophoblast is discontinuous over the decidual cells and the decidual cells participate in the wall of the trophoblast blood space (between arrows). Longitudinal section. Day 7, 2 P.M. Figure 7, x3,800. Figure 7a, ~ 2 6 , 2 0 0 . TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT Fig. 8. This scanning electron micrograph illustrates the trabecular nature of the trophoblast processes that extend from one side of the trophoblast blood space (TBS) to the other, connecting the regions of trophoblast that are juxtaposed to the decidual cells (Dec) and Reichert’s membrane. Parietal endoderm and Reichert’s membrane cannot 211 be seen in this micrograph because the trophoblast is folded back and covers them (arrows). In this micrograph the visceral endoderm is at the bottom WE). Figure 8a is a higher magnification of the blood space in Figure 8. Note the maternal blood cells in the trabecular meshwork. Longitudinal view. Day 8 , 2 P.M. Figure 8, X 1,750. Figure 8a, X5.700. 212 A.O. WELSH AND A.C. ENDERS Fig. 9. This scanning electron micrograph gives a polar view of the implantation chamber. The area is similar to that shown in the previous micrograph except the egg cylinder was lost during processing. The parietal endoderm (upper left) is clearly visible in Figure 9a. The shape of the trophoblast trabeculae seen in Figure 9a in conjunction with the information in Figure 8 suggests that the trabeculae have a mesometrial-antimesometrial orientation. Polar view. Day 8, 2 P.M. Figure 9, X 750. Figure 9a, ~ 2 , 9 0 0 . flanges that protrude into the blood spaces form incomplete septa and have maternal blood cells on both surfaces when viewed in transmission electron micrographs. Scanning electron micrographs give the impression that the flanges seen in thin sections are trabeculae that form a network that crosses the trophoblast blood space between the trophoblast covering Reichert’s membrane and the trophoblast juxtaposed to decidual cells (Figs. 8, 9). By day 8 trophoblast cells have grown to giant proportions. These cells are electron lucent and contain many free ribosomes and polyribosomes. Stacks of RER cisternae are abundant in some cells (Fig. 7) but scarce in others. When scarce, RER cisternae tend to be more vesicular. The mitochondria are also fairly electron lucent and have few cristae. There are numerous lipid droplets, and patches of intermediate filaments occur in the cytoplasm but are most frequently found at the apical border next to decidual cells. The nuclei are very large and irregular in outline. Nucleoli are well developed. By day 8 it appears that trophoblast cells are connected to other trophoblast cells primarily by desmosomes. There is little evidence of typical apical junctional complexes by the afternoon of day 8. The layer of polarized decidual cells juxtaposed to the residual basal lamina of the uterine lumina1 epithelium on day 7 is no longer seen by the afternoon of day 8. Instead, the cells along the inner wall of the implantation chamber are flattened, elongated along the meso- metrial-antimesometrial axis (Fig. 61, and have no apparent apical polarization; that is, they do not have flanges on the surface toward the trophoblast. Many decidual cells in proximity to the implantation chamber and trophoblast are degenerating (Figs. 6, 11, 12). Although there are variations between cells, there are many similarities in the morphology of their degeneration, which appears to be apoptotic rather than necrotic. Nuclear chromatin marginates into large dense masses, nuclei often become deeply indented and may fragment, and nucleoli appear as dispersed coarse granules (Fig. 11).Decidual cells lose contacts with adjacent cells and round up. The cytoplasm becomes condensed; that is, the organelles become densely compacted with little intervening material (Fig. 12). The RER often dilates, and the cells become smaller and often break up into small fragments. In many cases the condensing cells or cell fragments may contain nuclear fragments and usually a n abundance of intermediate filaments. When this degeneration occurs in the vicinity of trophoblast cells, as it usually does, processes from trophoblast cells engulf the dying cells and cell fragments (Fig. 12). Trophoblast cells sometimes engulf whole decidual cells as they round up and detach from adjacent cells. Trophoblast cells also phagocytize some erythrocytes but not others. In addition to these phagosomes, trophoblast cells also contain inclusions that resemble secondary lysosomes or phagolysosomes, but few dense bodies that might include a significant lysosomal population are TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT 213 way as to form a portion of the wall of the blood vessel p i g . 13). This participation of decidual cells occurs before and after maternal blood begins circulating in trophoblast blood channels. Endothelial cells that appear osmotically damaged are also fairly common. These cells are extremely pale, with dispersed organelles and damaged membranes. As trophoblast cells engulf decidual cell fragments, they occupy the area once occupied by decidual cells and hence become close to the maternal blood vessels. When connections are observed between maternal blood vessels and trophoblast blood spaces, they usually appear as a direct transition from trophoblast to endothelium (Fig. 14).Although decidual cells are not seen participating directly in this coupling, there are spaces between decidual cells that do contain erythrocytes and could presumably form blood channels. At the same time that maternal blood begins flowing in trophoblast blood spaces, maternal vessels open into the mesometrial uterine lumen (Fig. 6). The epithelium is sloughed into the lumen, and degenerating, necrotic, epithelia1 cells are found mixed with maternal blood cells. There is no trophoblast in this region at this time. DISCUSSION Fig. 10. In this light micrograph of a polar view of the implantation site the maternal blood vessels are all cut in cross section, a n indication that they have a mesometrial-antimesometrial orientation. The egg cylinder was lost during tissue processing. On the right, the trophoblast blood space is filled with maternal erythrocytes whereas, on the left, it is empty. Polar section. Day 8, 2 P.M. X 150. present. Although trophoblast cells phagocytize effete decidual cells and occasionally a n erythrocyte, maternal phagocytic cells do not appear to be activated. The blood vessels in the implantation region become oriented in a mesometrial-antimesometrial direction. This is especially apparent in polar views of the implantation site in which the vessels appear in cross section (Fig. 10). In the vicinity of the implantation chamber the blood vessels are composed of continuous, nonfenestrated endothelial cells, often without a clearly distinguishable basal lamina. These differ from the blood vessels in the deeper stroma and those of mature decidua (Welsh and Enders, 1985). Decidual cells are usually in close proximity to endothelial cells and there is a material of moderate electron density filling the space between them. It is common to find decidual cells that are in contact with endothelial cells, and they often become interposed between endothelial cells in such a The results reported here support the position that the uterus plays a n active role during embryo implantation and yolk sac placentation in the rat. Although trophoblast cells migrate to the maternal vessels that are the blood supply for the parietal wall of the yolk sac placenta, they appear to do so because degeneration of the decidualized stromal cells that intervene permits trophoblast migration to the maternal vessels. This is similar to the earlier situation when spontaneous or programmed cell death of the uterine luminal epithelium (El-Shershaby and Hinchliffe, 1975) permits trophoblast migration to the uterine luminal basal lamina. Kirby and Cowell (1968) articulated the position that trophoblast cells, while capable of phagocytizing degenerating decidual cells, might not be able to dispose of healthy decidual cells. They derived this assumption from work of Billington (1963) that indicated deciduomata break down in the central area first, then progressively toward the outer regions. They presented no morphological data to indicate the cellular basis of cellular degeneration in this model. There have been no thorough electron microscopic studies of trophoblast migration or invasion of decidual tissue during normal pregnancy in the rat. Several light microscopic reports have described the invasiveness of trophoblast in ectopic sites (Fawcett, 1950; Kirby, 1965; Porter, 1967). These studies have shown that mouse trophoblast is highly invasive of nonuterine tissues and uterine stroma during the estrus cycle, but not invasive of decidualized uterine stroma. There does not appear to be a consensus of opinion about the cellular aspects of invasion in ectopic sites, due, no doubt, to the limits of observation imposed by the methods used. Porter (19671, however, used ultrastructural analysis to report active trophoblast invasion of renal parenchyma and found that it was accompanied by little necrosis, but there was no mention made of phagocytosis of kidney tissue by trophoblast. Some investigators reported that trophoblast cells transplanted to ectopic sites formed a meshwork that was confluent with the blood vessels of the host (Fawcett, 1950; Porter, 1967). 214 A.O. WELSH AND A.C. ENDERS Fig. 11. Although decidual cells are usually detached from adjacent cells and have undergone some fragmentation before being surrounded by trophoblast processes, in this micrograph trophoblast processes (arrows) are surrounding a decidual cell (Dec) that is beginning fragmentation (upper right) but is still attached to the underlying cells. Notice the deep indentations of the nuclear membrane and the dispersed granular nucleolus. A trophoblast blood space ('I'BS) containing a maternal platelet is at the top of the micrograph. The decidual cells are elongated and contain an abundance of intermediate filaments. Longitudinal section. Day 8,8 P.M. x 14,800. The work presented here shows the initial degeneration of the antimesometrial decidua that is restricted to the area adjacent to trophoblast. The pattern of cellular degeneration is often similar to that described by Kerr et al. (19721, who proposed the term apoptosis to describe the controlled physiological deletion of cells during em- bryonic development. They delineated a two-part process that included first the formation of apoptotic bodies and second their phagocytosis and degeneration within nearby cells. They contrasted this to necrosis or nonphysiological cell death (Wyllie, 1981) caused by trauma or noxious substances. TROPHOBLAST, STROMA, AND YOLK SAC DEVELOPMENT Fig. 12. This micrograph illustrates the interface of trophoblast and degenerating decidual cells. Although the decidual cell (Dec) in the lower right is still attached to the underlying cells, it appears to be separating from them. At the left there are decidual cell fragments that have not been phagocytized while the center of the micrograph is occupied by fragments that have been phagocytized. In the upper left 215 is a trophoblast blood space CTBS). Much of the cytoplasm of the decidual cells and their fragments (pale areas) is occupied by intermediate filaments. Also some of the cytoplasmic fragments contain nuclear fragments while others do not. Apparent phagolysosomes (PL) are also present in the trophoblast. Longitudinal section. Day 8 , 8 P.M. ~9,180. 2 16 A.O. WELSH AND A.C. ENDERS Fig. 13. Decidual cells (Dec) often form part of the walls of maternal blood vessels (MBV) and appear to have some junctional attachment to endothelial cells (arrows).Day 8 , 2 P.M. ~ 2 3 , 3 5 0 . In a manner similar to other apoptotic cells, degenerating decidual cells separate from adjacent cells and, after condensation and fragmentation of the nucleus and cytoplasm, become small, dense bodies. The cytoplasmic fragments sometimes contain nuclear fragments, but other organelles within the fragments often appear normal. These free cytoplasmic bodies do not show ultrastructural evidence of degradation. Most decidual cell apoptotic bodies are found phagocytized by trophoblast cells although some are free in the intercellular space between decidua and trophoblast. Also, trophoblast cells sometimes begin phagocytizing individual decidual cells as they round up and detach from the adjoining decidual cells before there is any sign of fragmentation. After they are taken up by other cells, apoptotic bodies undergo degenerative changes that are independent of lysosomal enzyme action (Kerr et al., 1972). Lysosomal enzymes, however, are implicated in the final degradation of the phagosomal contents because preexisting secondary lysosomes of the ingesting cell are depleted and residual bodies are produced. Trophoblast cells that ingest decidual cell debris do not appear to contain a n extensive lysosomal system, as their hydrolase enzyme activity is not high (Christie, 1967). Since cell death in deciduoma occurs in the absence of trophoblast, one would expect either a maternal phagocytic response or some sort of autolytic system where the by-products would be expelled into the uterine lumen. Unfortunately, there is no ultrastructural information about this process. In the case of decidual cell breakdown, lysosomal as well as nonlysosomal events of cellular breakdown might occur outside of the influence of the ingesting cells. If decidual cells are the source of lysosomal enzymes that are responsible for the final stages of cell breakdown, decidual cell death would be autolytic a s well as apoptotic. Several in vitro studies have addressed the question of trophoblast invasiveness. Salomon and Sherman (1975) favored a cytolytic mechanism to explain the displacement of uterine cell monolayers by mouse trophoblast outgrowths. Glass et al. (1979) found no evidence TROPHOBLAST,STROMA, AND YOLK SAC DEVELOPMENT 217 Fig. 14. Trophoblast (Tr)appears to tap maternal capillaries (MVB) by first becoming part of the blood vessel wall (large arrows). It is not clear whether this occurs through a displacement of decidual cells or is a direct intervention on the part of trophoblast. In this micrograph decidual cell .fragments occur in trophoblast, and fragmenting decidual cells are seen adjoining trophoblast. In the lower part of the micrograph, decidual cells (Dec) are in very close proximity (arrows) to endothelial cells (Endo). Longitudinal section. Day 8,8 P.M. ~ 7 , 4 5 0 . of’cell lysis but proposed that a variety of cultured mouse cells retract upon contact with mouse trophoblast. In further studies (Kubo et al., 19811, they found that both attachment and outgrowth of mouse trophoblast could be reversibly inhibited by protease inhibitors. Mouse trophoblast was able to degrade complex radiolabeled extracellular matrix that contained glycoproteins, elastin, and collagen (Glass et al., 1983).Although this may be important in species where trophoblast is primarily interacting with uterine matrix, in the rat and mouse there is little extracellular matrix between decidual cells at the time trophoblast cells come into contact with uterine stroma. In these species interaction of trophoblast cells with extracellular matrix may play a secondary role to interactions between trophoblast and decidual cells. The degeneration of large numbers of decidual cells in the region juxtaposed to trophoblast appears to be a programmed and controlled form of cell death that allows remodeling of the implantation chamber without disrupting the growth and development of the embryo and the integrity of the uterus. The trophoblast acts on the decidualized stroma by phagocytizing cellular fragments and fragmenting cells. The decidual cell mem- branes do not appear to be damaged as would be expected if the trophoblast were cytolytic. In other tissues where apoptosis is observed, large numbers of cells can also be removed without disrupting the basic arrangement of tissues and their function Wyllie, 1981). Later in gestation, days 13-16, a massive degeneration of the remaining antimesometrial decidua and primary trophoblast giant cells occurs in the region adjacent to the reformed uterine lumen (Welsh and Enders, 1983, 1985), but this appears to be more necrotic than apoptotic. As the decidual cells lining the implantation chamber degenerate and are phagocytized by trophoblast, the latter comes closer to the stromal blood vessels. From the information presented in this study it appears that trophoblast somehow occupies the position held by endothelial cells, but it is not clear how trophoblast comes to occupy this position. Other studies have shown that decidual cell processes penetrate endothelial basal laminae (O’Shea et al., 1983; Welsh and Enders, 1985; Parr et al., 1986), and this study clearly shows decidual cells in conjunction with endothelial cells lining vessels prior to and during the time of blood circulation in trophoblast blood spaces. Presumably as these decidual cells degenerate trophoblast could occupy the space once held by 218 A.O. WELSH AND A.C. ENDERS decidual cells and thus tap maternal vessels. On the other hand, trophoblast might be capable of a more direct approach to maternal blood. The fate of endothelial cells after they come in contact with trophoblast is not clear. It appears that trophoblast can form junctions with adjacent endothelial cells, but how endothelial cells are disposed of is not obvious. There is no evidence of apoptosis of endothelial cells although in a few instances there appears to be necrosis; that is, swollen, pale cells with osmotic damage. Also, since the implantation chamber is rapidly increasing in size at this time, mitosis of endothelial cells and growth of blood vessels may not keep pace with changes in the decidua, and the endothelium could become discontinuous without extensive cell death. An incidental observation that argues against invasiveness of rat trophoblast in opening maternal vessels is found in the mesometrial part of the implantation chamber on day 8. There is no trophoblast in this area at this time, yet the uterine lumina1 epithelium and basal lamina degenerate and maternal vessels become open to the mesometrial portion of the implantation chamber. This occurs a t the same time that blood vessels are tapped by trophoblast in the antimesometrial area. Epithelial cell death in the mesometrial region differs from that in the region juxtaposed to trophoblast (unpublished observations). According to guidelines proposed by Kerr et al. (1972), the mesometrial epithelia1 cells appear necrotic whereas the epithelia1 cells in the region of trophoblast appear apoptotic. After the maternal vessels are breached, maternal blood can move into trophoblast blood spaces. The method of perfusion of the uterus used in this study demonstrates that there is circulation of blood through these spaces. Scanning and transmission electron micrographs indicate that the blood spaces are lined by trophoblast lying against Reichert’s membrane and in contact with decidual cells. The two layers of trophoblast cytoplasm are connected by trabeculae or flanges. These may give structural support, keeping the two layers of trophoblast from separating, and may direct blood flow. The arrangement of trophoblast along Reichert’s membrane suggests a potential path for substances moving between the maternal circulation and visceral endoderm. Trophoblast is characteristically very thin in this area with many fenestrations. Carpenter (1980) studied the permeability of the hamster parietal yolk sac placenta on day 8 of gestation using horseradish peroxidase (HRP) and found that trophoblast, Reichert’s membrane, and parietal endoderm were highly permeable to HRP. He also noted the appearance of both open and closed fenestrations in the trophoblast lining Reichert’s membrane. Jollie (1968) used ferritin and Thorotrast to study the permeability of the rat parietal yolk sac between days 12 and 22 of gestation. He found that the parietal yolk sac was a selectively permeable barrier to material entering the yolk sac cavity. While this manuscript was in preparation a study by Parr and Parr (1986) was published. They intravenously injected various macromolecular tracers to study the permeability of the early decidual cell layer that separates the yolk sac from maternal blood vessels. On days 6 and 7 they found that the permeability of the decidual cell layer decreased as the mass of the tracers increased. On day 9, however, the tracers had ready access to Reichert’s membrane and the yolk sac placenta although the permeability of the stromal vessels per se was decreased at this time. They noted that the decidual cell layer separating the maternal blood vessels and the yolk sac placenta was greatly reduced, and that there was maternal blood adjacent to Reichert’s membrane and in the uterine lumen mesometrial to the ectoplacental cone, but they did not mention mural trophoblast. They suggest that the tracers reach the implantation chamber from blood spaces adjacent to Reichert’s membrane or from blood in the space adjacent to the ectoplacental cone. The work presented in this paper illustrates the cellular basis for the observations of Parr and Parr (1986). The decidual cell layer is reduced by degeneration of the cells, probably apoptotic, and their phagocytosis by trophoblast cells. The maternal vessels are then tapped and maternal blood flows through the trophoblast blood spaces that are adjacent to Reichert’s membrane. Although this study focused primarily on the development of mural trophoblast, incidental observations suggest that there are also blood channels in the abembryonic trophoblast later on day 8 but these cells have no direct contact with maternal vessels. On the other hand, the superficial layer of polar trophoblast that is mesometrial to the ectoplacental cone does tap directly into maternal vessels channeling maternal blood into the blood spaces in the polar trophoblast. ACKNOWLEDGMENTS It is a pleasure to acknowledge the valuable discussions and support of Sandra Schlafke during the course of this investigation. This research was supported by grant HD 10342 from the National Institute of Child Health and Human Development. LITERATURE CITED Anderson, J.W. (1959) The placental barrier to gamma-globulinsin the rat. Anat. Rec., 204:403-429. 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