Transport of horseradish peroxidase across monkey trophoblastic epithelium in coated and uncoated vesicles.код для вставкиСкачать
THE ANATOMICAL RECORD 211:174-183 (1985) Transport of Horseradish Peroxidase Across Monkey Trophoblastic Epithelium in Coated and Uncoated Vesicles JEAN M.WILSON AND BARRY F. KING Department o f Human Anatomy, School of Medicine, University of California Davis, C A 95616 ABSTRACT This study used membranous chorion of the macaque monkey placenta to examine uptake and processing of exogenous proteins. Tissue was incubated with either cationic or anionic horseradish peroxidase. Incubation time was varied between 5-25 min to follow the endocytic pathways. In spite of some differences in binding, uptake and processing of the isozymes was similar. In the presence of tracers at 37°C both horseradish peroxidases were taken up in large (150-175) nm diameter) coated vesicles. In addition, coated tubules 300-400 nm in length and 50100 nm in diameter were seen in the apical cytoplasm. Studies using ruthenium red indicated that the coated tubules were derived from long coated invaginations of the free surface that pinch off into the apical cytoplasm. Often, the tubules bud off small (85-105 nm diameter) protein-filled coated vesicles which traversed the cytoplasm and fused with the basal-lateral plasma membrane. In other cases, the tubules or vesicles lost their clathrin coats and fused to form larger endocytic vesicles which later fused with phagolysosomes. After longer incubation, larger uncoated vesicles (endosomes)were seen releasing their contents at the basal-lateral membrane. These results suggest that multiple transport pathways exist in this epithelium. The first, involving only coated structures, may function to sort and concentrate specific ligands important for embryonic development. The second, involving the formation and translocation of large uncoated vesicles to the basal-lateral membrane, may also provide nutrients to the embryo. A third pathway directs the protein to phagolysosomes where it is presumably degraded. Degradation products could be used by the cells of the membranous chorion or may provide nutrients to the embryo. The membranous chorion of the macaque monkey placenta is composed of a columnar trophoblastic epithelium backed by a relatively avascular connective tissue (King, 1981).As a result of superficial implantation in the macaque monkey, the membranous chorion is exposed to the uterine lumen during early gestation. Morphological studies have suggested that this epithelium may be absorptive, possibly taking up macromolecules from the uterine lumen (King, 1981). The suggestion is also supported by the observations that similar cells in other species have been shown to have a n absorptive or phagocytic function (for references see Dantzer, 1982; Burton, 1982; King, 1982a,b; 1984). However, in virtually all cases there is no direct evidence delineating the intracellular pathway(s) by which material actually crosses these cells. In other absorptive epithelia, protein may be routed to lysosomes andor transported across the cell in clathrincoated or uncoated vesicles (Farquhar, 1983).Because of our interest in establishing the role of extraembryonic membranes in early development, we have studied the processing of absorbed protein in the trophoblastic epithelium of the monkey membranous chorion. We used two isozymes of horseradish peroxidase (HRP), acid 0 1985 ALAN R. LISS, INC. phosphatase (AcPase)cytochemistry, and ruthenium red staining to analyze the pathways of protein uptake and processing. In addition to a pathway that routes vesicles to phagolysosomes, we demonstrate two pathways of transepithelial transport. The first pathway involves exclusively clathrin-coated structures, while the second involves larger, uncoated endosomes. MATERIALS AND METHODS Membranous chorion of macaque monkey (Macaca mulatta and M. cynomolgus) placentas was obtained after cesarean section performed at 25 to 40 days of gestation. Tissue was dissected away from the placental disc and washed with cold Earle’s Balanced Salt Solution (Gibco Laboratories, Grand Island, NY). For binding studies, tissue was transferred to Minimal Essential Medium, pH 7.4 (Gibco), gassed with 0 2 C02(95%-5%) containing 2 mg/ml Type VI or Type VIII HRP (Sigma Chemical Co., St. Louis, MO) and 5 mg/ml bovine serum albumin (BSA; Sigma). Isoelectric focusing showed the Type VIII HRP (AHRP) had a PI of 4.5, with a minor band a t 3.5. The Type VI HRP (CHRP) had Received May 21, 1984; accepted August 16,1984 TRANSPORT OF PEROXIDASE ACROSS TROPHOBLAST 175 (Fig. 1).In addition, a population of moderately electrondense granules distributed throughout the apical cytoplasm was not reactive (Fig. 2). These granules ranged from 300 to 1,000 nm in diameter and were relatively homogeneous in content. The well-developed Golgi apparatus which surrounds the nucleus exhibited extensive AcPase activity (Fig. 3). Often, nearly all of the cisternae contained reaction product, although some cis cisternae remained unlabeled. Although some of the trans-most cisternae exhibited morphological characteristics of GERL (i.e., thickened membrane, endoplasmic reticulum with ribosomes on one membrane face), a majority did not have these attributes. Small 65-75 nm diameter coated vesicles containing reaction product were also seen in the Golgi region but were not observed in the apical portion of the cell or in contact with any endocytic vacuoles. There were also many small coated vesicles in the Golgi region that were not reactive for AcPase. Fixation in the presence of ruthenium red resulted in staining of the glycocalyx of the apical cell surface (Figs. 4-6). Coated pits and long coated invaginations of the cell surface were stained, with staining varying from heavy to very light (Figs. 4,5).Areas of glycocalyx which were opposite clathrin-coated areas often exhibited periodicity in staining (Fig. 4). Unstained coated vesicles and tubules were also seen in the apical cytoplasm (Fig. 6). The stain did not penetrate between the cells below the level of the tight junctions. The glycocalyx at the apical surface was most prominent in association with clathrin-coated areas of the membrane (Figs. 7-10). This clathrin coat is present on the cytoplasmic side of surface invaginations as well as on noninvaginated areas of the membrane (Fig. 10). Incubation of the tissue with HRP isozymes at 4°C demonstrated a difference in the absorption of the glycoproteins to the cell surface. CHRP was bound to the glycocalyx over the clathrin-coated areas, whereas uncoated membrane areas, including microvilli, were unlabelled (Fig. 7). AHRP was not absorbed to any area obviously associated with the free surface, although occasional “coated vesicular profiles,” presumably attached to the surface via a narrow neck, were labeled (Fig. 8). After a short incubation (5-10 min) at 37”C, both isozymes were seen a t the apical surface in clathrinRESULTS coated pits and vesicles measuring 150-175 nm in diThe ultrastructure of the membranous chorion of the ameter (Figs. 9, 16). There was some variability in the rhesus monkey placenta early in gestation has been absorption and uptake of both glycoproteins, since even described in detail elsewhere (King, 1981). Therefore, after the longest incubation (25 rnin), some heavily laonly those features pertinent to this study will be de- beled coated pits and vesicles were adjacent to others scribed, specifically the vacuolar apparatus which in- that were unlabeled (Fig. 10). In addition to coated vesicludes the endocytic complex and Golgi-lysosomal cles, other clathrin-coated structures with complex shapes were seen in the apical cytoplasm (Figs. 11-13, system. AcPase cytochemistry showed abundant deposition of 16). A majority of these structures contained reaction reaction product in large vacuoles and multivesicular product, although occasional profiles were observed bodies in the apical cytoplasm (Figs. 1, 2). These vacu- without reaction product. Of these complex structures, oles ranged from 0.5 to 2 pm in diameter and often had the most common was a dumbbell-shaped profile 300irregular shapes (Fig. 2). The basal portion of the cell 400 nm in length with rounded ends 85-105 nm in also contained AcPase-positive granules. However, they diameter (Figs. 11-13, 16). The morphology of these were often smaller and less abundant than in the apical structures included coated cylindrical profiles (Fig. 161, cytoplasm. Small, electron-lucent vacuoles approxi- more compact cylinders with bulbed ends (Fig. 111, and m-eatlv constricted at one end, mately 500 nm in diameter and a n extensive tubular- structures that were . , vesicular system which contained no reaction product forming a coated vesicular profile almost separated from were seen h the cortical portion of the apical cytoplasm the cylinder (Fig. 12). In some instances, the cylindrical a PI of 8.5-8.6, with a minor band at 9.0. BSA was included in all media because it resulted in improved morphology. Incubation was carried out for 5 min at 4°C. Tissue was fixed in 3% glutaraldehyde buffered with 0.1 sodium cacodylate-HC1at 4°C and processed as described below. For uptake studies, tissue was placed in medium identical to that of the binding studies but which was maintained at 37°C. Incubations were carried out for 5 to 25 min and then fixed as described above. All tissues were washed in 0.1 M cacodylate-HC1 buffer, pH 7.3, and incubated in a diaminobenzidine (DAB)medium which contained 0.5 mg/ml DAB (Sigma) + 0.01% H202 in phosphate buffer (pH 6.0). To allow penetration of substrate, samples were preincubated 15 min in buffer containing only DAB and then incubated 105 min in complete medium. Controls included incubation in DAB medium without H202 and incubation of tissue not exposed to tracer in complete medium. After DAB incubation, samples were postfixed in 2% OsO4 in 0.1 M collidine buffer, stained en bloc with 0.5% aqueous uranyl acetate, dehydrated through a series of cold acetones, and embedded in a n Epon-araldite mixture. For AcPase cytochemistry, tissue was immediately fixed (without incubation in tracer) in 2% paraformaldehyde + 2.5% glutaraldehyde or in 1.5% glutaraldehyde in 0.1 M cacodylate-HCl buffer, pH 7.3, a t 4°C for 1hr. Enzyme localization was carried out as previously described (King and Wilson, 1983)in a medium containing 40 mM Tris maleate + 11.5mM Na-b-glycerophosphate + 2.4 mM Pb(N03)2 + 5% sucrose. After incubation, tissue was washed in buffer, postfixed in 1% Os04 buffered with veronal acetate (pH 7.4) and stained en bloc with 0.5% uranyl acetate in Tris maleate buffer. Controls included incubation in the absence of substrate or heating the tissue to 60°C for 15 min prior to incubation in complete medium. For ruthenium red cytochemistry, tissue was fixed for 5 min at 4°C in 3% glutaraldehyde in 0.1 M cacodylateHC1 buffer (pH 7.3) and then immersed in a solution containing 0.5% ruthenium red (Polyscience, Inc., Warrington, PA), 2% glutaraldehyde, and 0.067 M cacodylate-HC1 buffer (pH 7.3) for 2 hr at 4°C. Subsequent processing was basically according to the method of Luft (1971). 176 J.M. WILSON AND B.F. KING Fig. 1. AcPase. Apical portion of epithelial cell. Several nonreactive vesicles of the tubulovesicular system are evident (asterisks), as well as a nonreactive multivescular body (MVB) and a large vacuole (which may be an MVB) filled with reaction product (top). ~20,900. Fig. 2. AcPase. Large (1.8 pm diameter) lysosome with dense reaction product. The cells also have a population of moderately electron-dense granules of various sizes that are unreactive for AcPase (asterisk) x 18,200. Fig. 3. AcPase. Extensive reaction product in Golgi cisternae. ~20,900 portion of the profile was uncoated on one or both sides; however, the rounded ends retained their clathrin coat (Figs. 13, 16). Coated vesicles 85-105 nm in diameter and filled with reaction product were seen in the apical cytoplasm (Fig. 16). However, they were distinct from another class of smaller (65-75 nm diameter) coated vesicles seen in both the apical cytoplasm and Golgi region but which did not contain reaction product (Fig. 16).After longer incubation, 85-105 nm diameter HRPfilled coated vesicles were seen below the level of the tight junction (Fig. 17) and fused with the basal-lateral plasma membrane (Fig. 18). In addition to coated structures, uncoated tubular profiles 200-400 nm in length and 50-100 nm in diameter and filled with reaction product were located near the apical membrane (Fig. 10). Uncoated tubules were also fused with endocytic vacuoles and phagolysosomes (Figs. 14, 15; see also Fig. 16, inset). Phagolysosomes were identified based on correspondence in size and distribution of AcPase-positive vacuoles (see Fig. 2) to similar vacuoles containing HRP reaction product in the uptake experiments. Smooth surfaced vesicles 140-175 nm in diameter, which contained a rim of reaction product, occurred after short incubation (Figs. 13, 16) and were often fused with larger endocytic vacuoles (Fig. 13).After longer incubation (15-25 min), the endocytic vacuoles surround large phagolysosomes and often fuse with them (Fig. 15). Large, smooth surfaced vesicles containing reaction product were present toward the basal aspect of the cell, and at 25 min of incubation some were fused with the basal plasma membrane (Figs. 19,ZO).The basal lamina contained reaction product in discrete areas that were associated with a recess in the basal membrane. The reaction product was most dense adjacent to the basal cell membrane (Fig. 20). Smooth surfaced vacuoles also were fused with the lateral membrane and reaction product was seen in punctate areas of the intercellular space, with adjacent unlabeled areas (not illustrated). There was no reaction product in the intercellular space in or near the tight junctions a t any time. HRP reaction product occurred in a few connective tissue cells near the epithelium after the longer incubations, but was not present after short incubations. Although the Golgi apparatus was well developed in these cells, HRP reaction product was not found in the cisternae or associated vesicles. In addition, the population of moderately electron-dense granules in the apical cytoplasm never contained reaction product, regardless of incubation time (Fig. 14). No differences in uptake or TRANSPORT OF PEROXIDASE ACROSS TROPHOBLAST 177 More importantly, it demonstrates two distinct pathways for vesicular transepithelial transport of the absorbed protein-one using coated vesicles and another using uncoated “endosomelike” vacuoles. Transepithelial transport via two distinct pathways is uncommon, as most epithelia use either coated vesicles or smooth vesicles to transport proteins across the cells (see below). The dual transport pathways may play a role in the nutrition and development of the embryo. Surface Binding Surface binding studies show adsorption of CHRP to the glycocalyx over clathrin-coated areas, whereas AHRP is seen only in very deep coated pits. Adsorption of CHRP to the glycocalyx may be mediated by the molecular charge of the protein. The cell surface is negatively charged, which can result in preferential adsorption of positively charge molecules to the glycocalyx (Mehrishi, 1972). If the binding is solely related to charge, the patchy distribution of the CHRP reaction product suggests that anionic sites are clustered over clathrin-coated areas of the membrane. This contrasts with macrophages, which diffusely bind positively charged molecules, including CHRP (Thyberg and Stenseth, 1981). The difference in distribution may reflect a functional difference, as the mechanism of internalization of cationic protein in macrophages involves large membrane folds rather than vesicles (Thyberg and Stenseth, 1981). The appearance of AHRP in the deep pits may be a result of incomplete washing of inaccessible pits, as binding of native (PI 6-7) HRP is morphologically undetectable in mouse peritoneal macrophages (Thyberg and Stenseth, 1981).However, charge may play a role in the adsorption of anionic macromolecules to the cell surface, as binding sites for negatively charged ligands have been demonstrated in macrophages (Brown et al., 1980). Also, other factors, such as carbohydrate residues, may play a role in adsorption of the glycoprotein to the cell (Ashwell and Harford, 1982). Carbohydrate-specific binding sites have been demonstrated (Kawasaki et al, 1978; Stahl et al., 1978), and HRP has been shown to bind in a specific and saturable manner to many cells Fig. 4. Ruthenium red. The glycocalyx of the apical surface is stained, (Straus, 1983). with staining in the coated pit exhibiting periodicity (arrows). x 63,600. One interesting observation was the presence of coated Fig. 5. Ruthenium red. This figure illustrates the variation in stain- pits with no detectable reaction product immediately ing intensity, which may indicate restricted access of the stain to some adjacent to a heavily labeled pit. This phenomenon has coated structures. The coated tubule (CT)exhibits heavy staining while also been reported in sea urchin egg-coated vesicles the coated pit (CP) is lightly stained. ~63,600. (Fisher and Rebhun, 1983). This observation may be Fig. 6. Ruthenium red. Coated tubule (CT) and vesicle (CV) have no explained by 1)heterogeneity of coated pits; 2) the empty staining of the glycocalyx, indicating their dissociation from the sur- pit may have a large neck out of the plane of section, face. ~ 6 3 , 6 0 0 . which allowed the HRP to be washed out; or 3) the glycocalyx binding sites may have been loaded with processing were present at difyerent gestational ages, endogenous protein. Further studies are needed to clarand controls showed no endogenous peroxidase activity ify this observation. in the cells. Uptake and Processing DISCUSSION We used two isozymes of HRP and AcPase and ruthenium red cytochemistry to attempt to reconstruct the pathways of endocytosis and processing of absorbed protein in the columnar epithelium of the monkey membranous chorion. This study confirms the suggestion that this trophoblastic epithelium is absorptive (King, 1981). Cells of the membranous chorion appear to take up and process both HRP isozymes by similar mechanisms, suggesting that charge does not play a role in their ultimate fate in this system. Only a few studies have compared uptake of isozymes of HRP. Endothelial cells in culture take up cationic HRP more rapidly than neutral or anionic HRP (Davies et al., 1981). Macrophages 178 J.M. WILSON AND B.F. KING Fig. 7. CHRP, 5 min at 4°C. Reaction product is concentrated in the glycocalyx over clathrin-coated areas. ~ 4 4 , 9 0 0 . Fig. 8. AHRP, 5 min at 4°C. The glycocalyx opposite some clathrincoated areas is unlabeled (arrowheads), hut an adjacent “deep-coated pit” is heavily labeled. ~ 4 5 , 0 0 0 . Fig. 9. AHRP, 10 min at 37°C. Coated pitivesicle filled with reaction product. Compare diameter with Figure 17. Adjacent unlabeled coated pithesicle is also apparent (arrow). Bar 200 nm. ~ 6 3 , 6 0 0 . labeled coated pit adjacent to unlabeled pits. Uncoated tubules ( U T i.e., lacking a clathrin coat) filled with reaction product are also shown. ~51,600. Fig. 11. AHRP, 25 min at 37°C. Coated tubule filled with reaction product. Note bulbous ends. The neck of the tubule near the end contains less reaction product, possibly preparing for fusion (arrow). ~63,750. Fig. 10. AHRP, 25 min at 37°C. The glycocalyx is most predominant opposite clathrin-coated areas (arrow).This figure also shows a heavily Fig. 12. CHRP, 10 min at 37°C. Coated tubule filled with reaction product, which seems to he concentrated in the bulbous termini of the tubule and one end (arrow) may he budding off. Compare diameter of bulbed end with Figure 17. Bar = 200 nm. X63,600. show little difference in amount of different HRP isozymes that are internalized, although the mechanism of uptake differs between isozymes and native HRP and AHRP are degraded more rapidly than CHRP (Thyberg and Stenseth, 1981; Stenseth et al., 1983) The limited number of studies comparing the uptake of HRP isozymes precludes generalizations about absorption rates and fate of internalized protein. The absence of uncoated invaginations a t the apical cell membrane suggests that protein uptake in this epithelium is mediated exclusively by coated vesicles and tubules. However, uncoated vesicles are observed in the apical cytoplasm, and AcPase cytochemistry indicates that these vacuoles are nonlysosomal. They probably correspond to endosomes or receptosomes found in many other cell types (Willingham and Pastan, 1980; Helenius TRANSPORT OF PEROXIDASE ACROSS TROPHOBLAST 179 Fig. 14. CHRP, 10 min at 37°C. This figure illustrates the continuity Fig. 13. AHRP, 10 min at 37°C. Tubule containing reaction product. The middle portion of the tubule has lost its clathrin coat (large arrow), of an uncoated tubule (UT) with a labeled endosome (El. Two moderbut the ends have retained it (small arrows). Small vesicles sometimes ately electron-dense granules which do not contain reaction product contained reaction product (arrowhead); alternatively, they may be a are also present (asterisks). x 63,600. cross section through a tubule. An uncoated endocytic vesicle (V) is fused with a labeled endosome (E). ~63,600. Fig. 15. AHRP, 15 min at 37°C. Apical portion of epithelial cell. Note microvilli (top). Large phagolysosome (PL) is surrounded by endosomes filled with reaction product. Both vesicular and tubular structures are fused with a phagolysosome (arrows). x 38,400. 180 J.M. WILSON AND B.F. KING et al., 1983).In this system, we define endosomes as acid hydrolase-negative vacuoles approximately 0.3-0.5 pm in diameter that contain HRP reaction product. Also included in this category are the acid hydrolase-negative tubules 200-400 nm in length and 50-100 nm in diameter. The endosomes may derive directly from coated pits which never leave the cell surface, as has been suggested to occur in certain fibroblasts (Willingham Fig. 16. AHRP, 10 min at 37°C. An example of several steps in the endocytic pathway. A large coated vesicle (CV) containing reaction product is seen near the surface, as well as uncoated vesicles (UV) of comparable diameter. Uncoated tubules (UT), partially coated tubules (PCT), and completely coated tubules (CT) loaded with reaction product and Pastan, 1983), or the coated pits may pinch off forming coated vesicles which subsequently lose their clathrin coat and fuse with one another. The 150-175 nm coated vesicles seen at the apical surface appear to be routed exclusively to endosomes, as coated vesicles of that diameter are never seen at the base of the cells. Another interesting “organelle” frequently observed in the present study is the “coated tubule.” The studies are also seen. An intermediate-sized coated vesicle (arrowhead) is contrasted with small (presumably golgi-derived) coated vesicles which contain no reaction product (arrows). A labeled phagolysosome (PL) is also apparelit. x 46,600. Inset, fusion of uncoated tubule with lysosome (arrow). x 36,400. TRANSPORT OF PEROXIDASE ACROSS TROPHOBLAST Fig. 17. AHRP, 25 min at 37°C. Intermediate-sized coated vesicle filled with reaction product near the lateral membrane at the base of the cell. Compare diameter with Figures 9 and 12. ICS, intercellular space. Bar = 200 nm. ~63,600. 181 Fig. 19. AHRP, 25 min at 37°C. Large uncoated vacuole filled with reaction product fused with basal cell membrane. BL, basal lamina. ~63,600. Fig. 18. AHRP, 25 min at 37°C. Apparent fusion of protein-filled coated vesicle (arrow)with lateral membrane. Intercellular space near point of fusion also contains reaction product, but more distal areas do not. ~63,600. Fig. 20. AHRP, 25 min at 37°C. Localized area of reaction product in a recess of the basal cell membrane and basal lamina (BL), suggesting a more advanced stage of vesicle fusion than that shown in Figure 19. Reaction product is absent in adjacent areas of BL and in deeper connective tissue. ~ 4 5 , 0 0 0 . using ruthenium red suggest that these structures are derived from elongated coated invaginations of the apical cell surface and a t least some of these structures are detached from the surface. The fate of the coated tubules appears to be varied. Although directionality is difficult to determine using static electron microscopic images, the fact that the bulbed ends of the coated tubules correspond in size to the protein-filled coated vesicles seen a t the base of the cells suggests that the termini of the tubules are the source of the 85-105 nm coated vesicles. These coated vesicles then traverse the cell and fuse with the basal-lateral membrane. A class of coated vesicles of this intermediate diameter has been described in rodent liver, and they may be a distinct shuttle compartment between the Golgi and plasma membrane (Croze et al., 1982).In the present study, the intermediate-sized vesicle may be involved in transport of protein across the epithelium. The observation that coated vesicles may be involved in transcellular movement of protein across trophoblastic cells of membranous chorion is consisent with similar conclusions in other absorptive epithelia (King and Enders, 1971; Rodewald, 1973; Moxon et al., 1976; Van Deurs et al., 1978; King, 1982a,b). A slightly different pathway was described in thyroid follicular cells by Herzog (1983). He observed coated pits at both the apical and basal-lateral cell membranes, but pro- posed an intermediate compartment of small uncoated vesicles. The presence of partially coated tubules suggests that coated tubules also give rise to the uncoated tubules seen in the apical cytoplasm. The uncoated tubules are nonlysosomal but are often fused with other endocytic vesicles. They may represent a n intracellular pathway leading to degradation of endocytosed ligand. Tubules that label with protein tracer have been reported in several epithelia (Graham and Karnovsky, 1966; King and Enders, 1970; Orlic and Lev, 1973) and tubular structures have been implicated as sites of intracellular sorting events in neonatal rat gut, guinea pig yolk sac, and liver (Abrahamson and Rodewald, 1981; King, 1982b; Geuze et al., 1983). In these systems the tubules were uncoated or had a n incomplete clathrin coat. In addition to transepithelial transport in intermediate-sized coated vesicles, protein accumulates in endosomes that migrate to the basal portion of the cell and release their contents. Although transport via this mechanism is not common, apparent endosomal transport of intact IgG was demonstrated in neonatal pig gut (Kraehenbuhl and Campiche, 19691, and IgA is transported across hepatocytes in small, uncoated vesicles (Reston et al., 1980). The movement of viral proteins from the apical cell membrane to the basal membrane J.M. WILSON AND B.F. KING 182 embryo. After crossing the membranous chorion, substances could gain access to the embryo by crossing the amnion or the yolk sac (King, 1980; King and Wilson, 1983). The coated tubule-coated vesicle pathway may function to sort and concentrate specific ligands important for embryonic development. The endosomal transport pathway may also provide nutrients to the embryo before the establishment of the definitive placenta and could function concurrently with the chorio-allantoic placenta. As in some other epithelia (Maunsbach, 1966; Van Deurs, 1978), the lysosomal system may have a major role in processing internalized protein in these trophoblastic cells. Endosomes fuse with phagolysosomes, presumably resulting in degradation of internalized protein. Low molecular weight degradation products may diffuse out of the lysosomes to provide nutrients to the rapidly growing, but relatively avascular, membranous chorion or may diffuse to the embryo. Since this trophoblastic epithelium is functionally active during the period of major organogenesis, teratogens gaining access to the uterine lumen could potentially reach the embryo via this route, resulting in malformation or embryonic death. ACKNOWLEDGMENTS Fig. 21. A summary of the pathways of protein uptake and processing in the membranous chorion. Large (150--175nm diameter) coated vesicles lose their coats and form endosomes (pathway 1).The endosomes then fuse with phagolysosomes (pathway la) or fuse with the basallateral membrane (pathway lb). An alternative pathway involves the formation of coated tubules (pathway 2). The coated tubules bud off intermediate-sized (85-105 nm diameter) coated vesicles that fuse with the basal-lateral membrane (pathway 2a). These medium-sized coated vesicles are distinct from the small (65-75 nm diameter) coated vesicles that are derived from the Golgi apparatus. The portion of the coated tubule that remains loses its coat and fuses with endosomes or phagolysosomes (pathway 2b). in endosomes and nonlysosomal multivesicular bodies has also been demonstrated (Matlin et al., 1983). This study demonstrates the previously unreported phenomenon of coated vesicle and endosomal diacytosis operating in tandem. The multiple transport pathways present in these cells are summarized in Figure 21. Since these studies were carried out in vitro, some of the absorbed protein, or protein that was localized in the intercellular spaces or basal lamina, may have diffused in from the cut edges of the tissue or from the connective tissue side of the sample. However, in both the case of coated vesicle transport and endosomal transport, the patchy distribution of reaction product in the intercellular space or basal lamina, which is usually associated with a recess in the plasma membrane, indicates vesicular transport and release rather than a type of diffusion artifact. Developmental Significance The transport of proteins or other substances across the membranous chorion may have important implications for the normal growth and development of the This investigation was supported by National Institutes of Health grants HD 11658 and RR 00169. We wish to thank Dr. Andrew G. Hendrickx and the staff of the Perinatal Biology Unit of the California Primate Research Center for assistance in obtaining the tissue; the laboratory of Dr. Jerry Hedrick, Department of Biochemistry and Biophysics, for determining the isoelectric points of the isozymes; and Ms. Katherine Wilson for preparing the drawing. J.M.W. was supported by NIH training grant HD 07131 through a portion of this investigation. LITERATURE CITED Abrahamson, D.R., and R. Rodewald (1981) Evidence for sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine. J. Cell Biol., 91t270-280. Aswell, G., and J. Harford (1982)Carbohydrate-specific receptors of the liver. Ann. Rev. Biochem., 51531-554. Brown, M.S., S.K. Basu, J.R. Falck, Y.K. Ho, and J.L. Goldstein (1980) The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages. J. Supramol. Struct. 13r67-81. 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