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/ . Embryol. exp. Morph. Vol 24, 2, pp. 313-330, 1970 313 Printed in Great Britain Protein transmission across the rabbit foetal membranes By A. E. WILD 1 From the Department of Zoology, University of Southampton SUMMARY Fluorescent protein tracing, involving F.I.T.C.-conjugated proteins and the fluorescent antibody technique, was employed to study the sites and mechanism of transport of a variety of proteins across the rabbit foetal membranes. No evidence was found for an intercellular transmission of proteins across the yolk sac splanchnopleure to the exocoel, but all proteins were shown to become localized in absorptive vesicles in the yolk-sac endoderm. Different proteins became similarly localized in absorptive vesicles having different sizes and profiles. Characteristic broken vesicles were present, and more than one protein was demonstrated in each absorptive vesicle. The yolk-sac endoderm was confirmed as the selective site for transmission of proteins to the foetal circulation, since only proteins readily detected in the foetal serum were present in, and below, the basement membrane. The paraplacental chorion was shown to be the site for transmission of proteins to the exocoel and the process to be one of diffusion. Unlike normal proteins, F.l.T.C. conjugates readily became localized within macrophages present in the paraplacental chorion and yolk-sac vascular mesenchyme. These findings are discussed in the light of differences previously shown to occur between transmission of proteins to the foetal fluids and to the foetal blood, and in the light of a current hypothesis to account for selection of proteins by the yolk-sac endoderm. INTRODUCTION In a previous study of the protein composition of the rabbit foetal fluids (Wild, 1965) a paucity of the high molecular weight proteins was found in the exocoelomic, amniotic and allantoic fluids. A greater proportion of albumin was also found in these fluids when comparison was made with the foetal and maternal sera. These findings pointed to a selective transmission of proteins based on molecular size and similar findings for the amniotic fluid of other species are consistent with this view (for details see review by Wild, 1966). In contrast, transmission of proteins to the foetal rabbit circulation is clearly independent of molecular size since the process favours homologous y-globulin (Hemmings & Brambell, 1961) and even 19 S y-globulins are transmitted (Hemmings & Jones, 1962; Kaplan, Catsoulis & Franklin, 1965). A further 1 Author's address: Department of Zoology, The University of Southampton, Southampton, Hants, SO9 5NH, England. 314 A. E. WILD interesting difference is that y-globulins of different species are transmitted selectively to the foetal circulation but non-selectively to the fluids (Batty, Brambell, Hemmings & Oakley, 1954; Kulangara & Schechtman, 1962). Whilst no doubt exists that the yolk-sac splanchnopleure is the site for transmission of proteins to the foetal circulation (Brambell, Hemmings & Henderson, 1951) the site for transmission to the exocoel has been a matter for more speculation. Kulangara & Schechtman (1962) suggested that proteins reached the exocoel by diffusion across the yolk sac, which acted as a molecular sieve. Brambell et al. (1951) could not rule out the paraplacental chorion, and I have also emphasized its possible role since in in vitro dialysis experiments it was much more permeable to protein than the yolk sac (Wild, 1965). In the present investigation use has been made of fluorescent protein tracing in order to locate the site and elucidate the mechanism of transmission to the exocoel in vivo, and also to throw light on the selective mechanism involved in transfer to the foetal vitelline circulation. MATERIALS AND METHODS Direct labelling of a variety of proteins including rabbit y-globulins (98 % pure, Mann Laboratories), human y-globulins (98% pure, Calbiochem), bovine y-globulins (Armour, Fraction 11), human serum albumin (Mann Laboratories), and egg albumin (Armour) was carried out according to Nairn (1964) except that fluorescein isothiocyanate (F.I.T.C.) adsorbed on to celite (Calbiochem) was used. Conjugation of proteins at a ratio of 20 mg protein: lmg F.I.T.C. was continued overnight at 4 °C. Non-reacted F.I.T.C. was removed by Sephadex G-25 chromatography and the conjugates brought back to their original volumes by ultrafiltration in Visking tubing. Final concentrations of albumins were 30 mg/ml and y-globulins 10 mg/ml. For the fluorescent antibody technique, ammonium-sulphate-fractionated, high titre rabbit antisera to human and bovine y-globulin, and to egg albumin, were conjugated to F.I.T.C. on celite, and in the case of human y-globulin, also to Lissamine rhodamine R.B. 200 chloride (R.B. 200 Cl) as described by Nairn (1964). Non-reacted fluorochromes were removed as previously described. F.I.T.C.-labelled rabbit anti-human a-2 macroglobulin and R.B. 200 Cl labelled anti-human serum albumin were obtained commercially (Mann Laboratories). Immunoelectrophoresis employing the conjugated absorbed antisera revealed only single lines for all antigens. In the case of the y-globulins this was yG. For direct tracing, protein conjugates were sterilized by filtration and after laparotomy injected into the uterine lumen of Dutch rabbits, 26-28 days pregnant, at a dose of 1 ml/conceptus. At various time intervals, ranging from 15 min to 8 h, rabbits were killed with nembutal and the conceptuses exposed. After washing with normal saline, areas of yolk sac, paraplacental chorion and amnion were removed, spread over rectangular windows cut in card, and fixed Protein transmission 315 for 24 h in 10% formol/saline or 95% ethanol. Where appropriate, exocoelomic and amniotic fluids and foetal blood were collected and tested for the presence of heterologous proteins by means of the interfacial ring test and in some cases immunoelectrophoresis. Subsequent dehydration, clearing and embedding of the membranes was carried out according to Nairn (1964). After dewaxing in three changes of xylene, sections (4 /t) were passed through three changes of 100% ethanol and then three changes of phosphate-buffered saline (0-01 M, pH 7-2), and then mounted in a medium consisting of nine parts glycerol/one part phosphate-buffered saline. Membranes from conceptuses of non-injected horns, similarly processed, were used as controls. Sections were examined for u.v. fluorescence in a Wild M 20 microscope. Photographs were taken in colour using Kodak High Speed Ektachrome and in black and white using Tri-X. For tracing by the fluorescent antibody technique, normal human serum was used as a source of human y-globulin, a-2 macroglobulin and albumin, and was supplemented with egg albumin at a concentration of 30 mg/ml. Solutions containing human and bovine y-globulins in normal saline (each at 10 mg/ml) were also used. Injection and subsequent procedures were as for direct tracing, except that membranes were fixed in cold 95% ethanol and processed according to the technique described by Sainte-Marie (1962). In order to reduce nonspecific staining, conjugated antisera were absorbed first with pig liver powder (Burroughs Wellcome) followed by one or sometimes two absorptions with foetal membrane powder prepared from chorion, yolk sac and amnion of conceptuses not exposed to heterologous proteins. Rabbit antibovine y-globulin serum was found to cross-react slightly with human y-globulin, and to remove such cross-reacting antibodies, 5 ml batches of conjugated antiserum were absorbed with 50 mg human y-globulin for 24 h at 4 °C. The precipitate was then removed by filtration. Absorbed, specific conjugated antisera were applied to dewaxed sections for 30 min in a saturated environment. Sections were then washed for 1 h in two changes of phosphate-buffered saline. Blocking tests were carried out with non-conjugated antisera but the best controls were sections of membranes not exposed to antigen and which had been similarly processed. RESULTS Direct tracing with protein F.T.T.C. conjugates All protein conjugates, as indicated by specific fluorescence, became localized in vesicles in the endodermal cells of the yolk-sac splanchnopleure (Figs. 1, 2). There was no indication that conjugates passed through, or were discharged into, intercellular spaces. In some experiments, particularly short-term ones, fluorescence could not be detected in any of the sections of yolk sac examined, or it was absent from certain areas of the yolk sac, thus giving identical appearance to control tissue. In such cases the conjugates had presumably failed to spread around the conceptus, or had done so unevenly. 316 A. E. W I L D VHS A Protein transmission 317 The vesicles showed a variation in size and in distribution and intensity of fluorescence. After 15 and 30min exposure to conjugates, the vesicles were mainly sub-apically located (Fig. 1A, B) and then became more randomly distributed (Fig. 1C-E; Fig. 2A, C). Fluorescence was distributed throughout vesicles, or it was confined to the perimeter, or unevenly distributed on the perimeter of intact and broken vesicles (Fig. 1B, E; Fig. 2 A, C). Broken vesicles were the largest and usually confined to the supra-nuclear region, whilst smaller vesicles were more randomly distributed. Bovine, human, and rabbit y-globulin, human serum and egg albumin conjugates, all showed a similar localization of fluorescence in vesicles which were similarly distributed within the endodermal cell. However, only with human and rabbit y-globulin conjugates was fluorescence ever detected in the basement membrane of the yolk-sac endoderm, in the vascular mesenchyme, or in the lumen of the vitelline vessels (Fig. 1D, E; Fig. 2 A, B). Vesicles containing rabbit ABBREVIATIONS ON FIGURES bb = brush border; bm = basement membrane; bv = broken vesicle; cs = canalicular system; cyt = cytotrophoblast; e = erythrocyte; em = exocoelomic mesothelium; end = endothelium; Imt = loose mesenchymal tissue; m — macrophage; // = nucleus; tgc = trophoblastic giant cell; vm = vascular mesenchyme; vv = vitelline vessel; yse = yolk-sac endoderm. Fig. 1. (A) Yolk-sac splanchnopleure of rabbit no. 20 exposed to rabbit y-globulin F.l.T.C. conjugate for 30 min. The conjugate is confined to vesicles situated mainly in the apical regions of the yolk-sac endoderm. x 100. Bright-field fluorescence 1-^ min exposure. (B) Another area of yolk-sac splanchnopleure of rabbit no. 20 at higher magnification. The conjugate is confined to intact and broken vesicles. Erythrocytes in a vitelline vessel show autofluorescence. x 500. Dark-field fluorescence, l^min exposure. (C) Yolk-sac splanchnopleure of rabbit no. 7 exposed to bovine y-globulin F.l.T.C. conjugate for 2i h. Vesicles containing conjugate are distributed throughout endodermal cells. The conjugate is absent from the basement membrane but occurs in a short region of the exocoelomic mesothelium (arrowed) indicating derivation from the exocoel. x 100. Bright-field fluorescence, \\ min exposure. (D) Yolk-sac splanchnopleure of rabbit no. 48 exposed to human y-globulin F.l.T.C. conjugate for 2^h. Note the presence of the conjugate in the basement membrane and in macrophages in the vascular mesenchyme. x 100. Bright-field fluorescence, J£ min exposure. (E) An area of yolk-sac splanchnopleure of rabbit no. 48 at higher magnification. Note the variation of size of vesicles containing the conjugate in the endoderm. The largest are broken and fluorescence is less intense in these. A vesicle (arrowed) appears to lie within the basement membrane, which shows a diffuse distribution of the conjugate. Other vesicles lie in close apposition to the basement membrane. Macrophages containing small, intensely fluorescent vesicles lie close to vitelline vessels. Specific fluorescence indicative of the conjugate is present along the exocoelomic mesothelial border, again indicating a derivation from the exocoel. x 500. Dark-field fluorescence, !•£• min exposure. 318 A. E. WILD Protein transmission 319 and human y-globulin were often seen closely apposed to the basement membrane and on rare occasions apparently within it (Fig. 1E), but whilst fusion of vesicles with, or discharge of their contents into the basement membrane seems the only way of accounting for the observed specific fluorescence, more convincing evidence of such a process was not observed. Diffuse specific fluorescence was also evident in the vascular mesenchyme and in the endothelium of the vitelline vessels, and where red blood cells remained in the vessel lumen, it was frequently seen around them (Fig. 2 A). A further striking feature with rabbit and human y-globulin conjugates was their localization in macrophages which frequently bordered the vitelline vessels (Fig. I D , E) or lay in the vascular mesenchyme in close apposition to the basement membrane (Fig. 2A, B). Within the macrophages fluorescence was confined to much smaller vesicles than observed in the endodermal cells. All conjugates were unevenly distributed along the exocoelomic mesothelium (see Fig. 1C, E) and at least in the case of bovine y-globulin, human and egg albumin, this represented a derivation from the exocoelomic fluid. Absence of fluorescence indicative of bovine y-globulin, human and egg Fig. 2. (A) Another area of yolk-sac splanchnopleure of rabbit no. 48 (cf. Fig. 1 D, E) showing presence of the conjugate around erythrocytes in a vitelline vessel. A macrophage lies in close contact with the basement membrane. Diffuse fluorescence is present along the brush border of the yolk-sac endoderm. x 500. Dark-field fluorescence, \% min exposure. (B) Yolk-sac splanchnopleure of rabbit no. 17 exposed to rabbit y-globulin F.I.T.C. conjugate for 5 h. Vesicles are few in number in the yolk-sac endoderm but the basement membrane and underlying macrophages show presence of the conjugate, which is also dispersed through the vascular mesenchyme. x 400. Bright-field fluorescence, 1-^min exposure. (C) An area of yolk-sac splanchnopleure of rabbit no. 48 in which broken vesicles are particularly prominent but conjugate is absent from the basement membrane and vascular mesenchyme. Note the variation in intensity of fluorescence within the vesicles. x400. Bright-field fluorescence, 1-J-min exposure. (D) Paraplacental chorion of rabbit no. 7 exposed to bovine y-globulin F.I.T.C. conjugate for 2\ h. In this field two macrophages, containing numerous, brightly fluorescing droplets of conjugate, can be seen in the loose mesenchymal tissue. The basement membrane of the cytotrophoblast and the mesenchymal tissue itself show much weaker fluorescence. x400. Bright-field fluorescence, l^min exposure. (E) Paraplacental chorion of rabbit no. 5 exposed to human y-globulin F.I.T.C. conjugate for 30 min. The conjugate is localized in the basement membrane of the cytotrophoblast and dispersed throughout the loose mesenchymal tissue and exocoelomic mesothelium. Specificfluorescencealso occurs around the cytotrophoblast cells in some areas, but not within them, x 100. Bright-field fluorescence, H min exposure. (F) Paraplacental chorion of rabbit no. 34 exposed to egg albumin F.I.T.C. conjugate for 3 h. In this field the conjugate is heavily localized in the cellular debris and surrounds giant cells in an area of giant-cell proliferation. There has been little penetration beyond the cytotrophoblast basement membrane, x 100. Bright-field fluorescence, H min exposure. 320 A. E. WILD Protein transmission 321 albumin from in and below the basement membrane was generally correlated with negative precipitin tests for these proteins in the foetal blood, but egg albumin was sometimes detected in low titre when there was no evidence for its transmission to the vitelline vessels. However, all conjugates were readily detected in the exocoelomic fluid and within 15min of injection in the case of that conceptus nearest the injection site. Entry into the exocoel was clearly by way of the paraplacental chorion, as evidenced by diffuse fluorescence around the trophoblastic giant cells and cytotrophoblast, and by intense fluorescence of the basement membrane of the cytotrophoblast and of the loose mesenchymal tissue and exocoelomic mesothelium (Fig. 2E, F). All conjugates, again in the form of small vesicles, were found in macrophages dispersed through the mesenchymal tissue (Fig. 2D). Such macrophages varied considerably in density throughout the chorion, but they were more abundant in longer term experiments and easily observed in living tissue spread as a thin sheet and exposed to u.v. light (Fig. 3 A). Diffuse specific fluorescence was also observed in the extracellular ground substance and on the myofibrils present in the amnion. Again in longer term experiments (after 2 h), all conjugates were seen as fluorescent droplets in the amniotic epithelium and mesenchymal tissue (Fig. 3B), and readily observed in spreads of living amnion. Because egg albumin was sometimes detected in the foetal serum when specific fluorescence was absent from the basement membrane and vitelline Fig. 3. (A) Spread of living chorion from rabbit no. 34 showing an area rich in macrophages in which the conjugate has become localized. Focused through the exocoelomic mesothelium. x 100. Bright-field fluorescence, J^min exposure. (B) Amnion of rabbit no. 34 showing intracellular localization of conjugate, x 100. Bright-field fluorescence, 1-fc min exposure. (C) Yolk-sac splanchnopleure of rabbit no. 40 exposed for 15 min to normal human serum supplemented with egg albumin, and treated with rabbit antihuman y-globulin F.l.T.C.-conjugated antiserum. Specific fluorescence indicative of human y-globulin is confined to the brush border and subapical vesicles in the endoderm. Fluorescence is also present along the exocoelomic mesothelium indicating protein derived from the exocoel. x 100. Bright-field fluorescence, \% min exposure. (D) Yolk-sac splanchnopleure of rabbit no. 40 treated with rabbit anti-egg albumin F.l.T.C.-conjugated antiserum. Weak diffuse specific fluorescence indicative of protein is present in the subapical canalicular system and concentrated below this in terminal absorptive vesicles, x 500. Dark-field fluorescence, \\ min exposure. (E) Yolk-sac splanchnopleure of rabbit no. 40 treated with rabbit antihuman y-globulin F.I.T.C. conjugate. Distribution of protein is similar to that seen in (D), but protein is additionally present on the brush border. x400. Bright-field fluorescence, \\ min exposure. (F) Yolk-sac splanchnopleure of rabbit no. 39 exposed for 30 min to normal human serum supplemented with egg albumin, and treated with rabbit antihuman y-globulin F.l.T.C.-conjugated antiserum. Protein is present in intact and broken vesicles, and on the surface of erythrocytes (arrowed). The basement membrane shows no specificfluorescencealthough vesicles lie in close apposition to it. x 500. Dark-field fluorescence, \\ min exposure. 322 A. E. W I L D Protein transmission 323 vessels, the possibility that protein and fluorescein became dissociated as a result of enzymic degradation within the vesicles, had to be considered. When F.I.T.C. alone was injected into the uterine lumen at a concentration of 15mg/ml in phosphate-buffered saline, and the foetal membranes examined after 3 h exposure, fluorescence was again detected in vesicles in the yolk-sac endoderm. These vesicles were much smaller than those observed with the conjugates and there was no fluorescence in or below the basement membrane. In the chorion bright diffuse fluorescence was distributed throughout the tissue, but no macrophages were located. Sections of yolk sac exposed to egg albumin conjugates were also treated with rabbit anti-egg albumin F.I.T.C. This treatment failed to reveal any further localization of fluorescence, either in or below the basement membrane, or in the vitelline vessels. In order to check whether the fluorescence observed in the basement membrane, the vascular mesenchyme and macrophages, was in fact human y-globulin conjugate and not free fluorescein, sections of yolk sac exposed to human y-globulin conjugate for 2\ h were treated with rabbit antihuman y-globulin R.B. 200 Cl. This resulted in a yellowish fluorescence in previously apple-green fluorescing sites and as opposed to orange-red fluorescence in yolk sac exposed to normal human y-globulin, indicating that human y-globulin was present as antigen in these sites. Indirect tracing with the fluorescent antibody technique All protein antigens (human serum albumin, a-2 macroglobulin and y-globulin; bovine y-globulin and egg albumin) were again detected within vesicles in the yolk-sac endoderm, as evidenced by specific fluorescence after treatment of sections with specific conjugated antisera (see Figs. 3-5). As with the direct tracing technique, only a non-selective intracellular route of entry was indicated. This was well illustrated in experiments of 15min duration, after which time proteins were localized on the brush border, in the subapical region, Fig. 4. (A) Yolk-sac splanchnopleure of rabbit no. 37 exposed for 5 h to normal human serum supplemented with egg albumin and treated with rabbit anti-egg albumin F.I.T.C.-conjugated antiserum. Note the variation in distribution and intensity of fluorescence within the vesicles. Many lie close to the basement membrane which shows no specific fluorescence, x 500. Dark-field fluorescence, 1$ min exposure. (B) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihuman y-globulin F.I.T.C.-conjugated antiserum. Note that the vesicles show a similar variation in intensity and distribution offluorescencewhen compared with those in Fig. 4 (A), indicating a similar distribution of the two proteins in the endoderm. The basement membrane, vascular mesenchyme and endothelium all show intense specific fluorescence, x 400. Bright-field fluorescence, 1^- min exposure. (C) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihuman y-globulin F.I.T.C.-conjugated antiserum. In this field broken vesicles are more evident and intense specific fluorescence indicative of human y-globulin is present within a vessel. The basement membrane shows no specific fluorescence in this region, x 500. Dark-field fluorescence, 1 | min exposure. 21 EMB24 324 A. E. WILD and concentrated immediately below this into vesicles (Fig. 3C-E). Greater non-specific fluorescence was observed with this technique, especially in nuclei and in erythrocytes, but specific fluorescence within vesicles was never observed in treated control tissue and blocking tests carried out with non-conjugated antisera considerably reduced specific fluorescence in experimental material. Distribution of fluorescence within vesicles was similar to that encountered with the protein F.I.T.C. conjugates, except that broken vesicles, although present, were not so clearly defined (Fig. 3F; Fig. 4C). Of those proteins investigated, Protein transmission 325 human y-globulin was the only one readily detected in the basement membrane, the vascular mesenchyme, and the vitelline vessels (Fig. 3F; Fig. 4B, C; Fig. 5 C, E) and could be located in such sites as early as \ h after exposure of membranes to antigen. Human serum albumin could also be detected in these sites, but only after longer exposure and with much less intensity of specific fluorescence. Although vesicles containing egg albumin and bovine y-globulin lay close to the basement membrane (Fig. 4A; Fig. 5B, D) they were never detected in or below it, but as in previous long-term experiments with conjugate, egg albumin was sometimes detected in low titre in the foetal serum. Except for bovine y-globulin, normal proteins were never observed in macrophages, either in the vascular mesenchyme or in the paraplacental chorion, and this provided the only striking difference between treatment of conjugated and normal proteins. Bovine y-globulin was sometimes observed in macrophages in the paraplacental chorion (Fig. 5D) and was present as droplets in mesenchymal cells of the amnion. After 15 min exposure, proteins were again detected in the exocoelomic fluid when they were absent from the foetal serum. Results illustrating this in a typical experiment are shown in Table 1. This rapid entry of proteins to the exocoel was again correlated with the presence of specific fluorescence indicative of all proteins (except human a-2 macroglobulin) in and around multinucleate giant cells, around the cytotrophoblast and in its basement membrane, and dispersed through the loose mesenchymal tissue and exocoelomic mesothelium (Fig. 5 A). Comparison of litres of bovine and human y-globulin Fig. 5. (A) Paraplacental chorion of rabbit no. 40 exposed for 15 min to normal human serum supplemented with egg albumin and treated with rabbit antihuman y-globulin F.I.T.C.-conjugated antiserum. Specific fluorescence is present in debris surrounding giant cells, in the cytotrophoblast basement membrane and dispersed throughout the loose mesenchymal tissue. Nuclei of cytotrophoblast and giant cells show non-specific fluorescence (arrow), x 100. Bright-field fluorescence, l^min exposure. (B) Yolk-sac splanchnopleure of rabbit no. 37 exposed for 5 h to normal human serum supplemented with egg albumin and treated with rabbit anti-egg albumin F.i.T.C.-conjugated antiserum. Specific fluorescence is confined to vesicles in the yolk-sac endoderm. Nuclei in the vascular mesenchyme (arrowed) show non-specific fluorescence, x 100. Bright-field fluorescence, 1^ min exposure. (C) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihuman y-globulin F.T.T.C.-conjugated antiserum. Note intense specific fluorescence in the vascular mesenchyme and lumen of vitelline vessel, x 100. Bright-field fluorescence, \\ min exposure. (D) Yolk-sac splanchnopleure and paraplacental chorion of rabbit no. 51 exposed for 3 h to a mixture of human and bovine y-globulins and treated with rabbit antibovine y-globulin F.i.T.C.-conjugated antiserum. Specific fluorescence is confined to vesicles in the yolk-sac endoderm and in the chorion to the loose mesenchymal tissue and macrophages. x 100. Bright-field fluorescence, 1^ min exposure. (E) Yolk-sac splanchnopleure of rabbit no. 51 treated with rabbit antihuman y-globulin F.i.T.C.-conjugated antiserum. Note intense specific fluorescence in the basement membrane and vascular mesenchyme. x 100. Bright-field fluorescence, l^min exposure. 326 A. E. WILD in the exocoelomic and amniotic fluids with foetal serum titres, confirmed the non-selective entry of these proteins to the fluids. Results for a typical experiment are shown in Table 2. Specific fluorescence representing protein derived from the exocoel was again evident in intermittent areas along the exocoelomic mesothelium of the yolk sac (Fig. 3C). Table 1. Transmission of human y-globulin and egg albumin to the fluids and blood of the single conceptus of rabbit no. 40,15 min after exposing it to 1 ml of normal human serum supplemented with 3% egg albumin Foetal fluid Exocoelomic Amniotic Serum Injected serum Titre against rabbit anti-egg albumin 128 32 — ve 1048000 Titre against rabbit anti-human y-globulin 8 — ve — ve 32000 Table 2. Transmission of human and bovine y-globulin to the fluids of the first conceptus of rabbit no. 51, 3 h after exposing it to a mixture of both proteins Foetal fluid Exocoelomic Amniotic Serum Injected solution Titre against rabbit anti-bovine y-globulin Titre against rabbit anti-human y-globulin 64 32 — ve 8000 64 32 32 8000 In order to determine whether or not human y-globulin and egg albumin were localized in the same vesicles, sections of yolk sac exposed to human serum supplemented with egg albumin were treated first with F.I.T.C.-labelled antiserum to egg albumin and the fluorescence of the same fields compared after subsequent treatment with R.B. 200 Cl-labelled antiserum to human y-globulin. Vesicles previously fluorescing apple green, gave an intermediate orange-yellow fluorescence after such treatment, and no fields were found in which apple-green fluorescing vesicles co-existed with reddish orange vesicles. Such results were interpreted as indicating that all vesicles contained both proteins. DISCUSSION The use of F.I.T.C. conjugates for tracing proteins presents certain limitations not encountered with the fluorescent antibody technique. The fluorochrome may alter the way in which protein is taken up into the cell and it may then Protein transmission 327 become dissociated from protein as a result of enzymic action. The fluorescent antibody technique, on the other hand, presents problems not encountered with F.l.T.C. conjugates, namely possible loss of antigenicity and non-specific staining. Thus findings common to both techniques are better guides to the mechanisms involved in uptake and transport of proteins than are findings attributable to one technique alone. All proteins, whether detected as conjugates or by fluorescent antibodies, were taken up intracellularly by the yolk-sac endoderm. This finding gives a visual demonstration that protein uptake is non-selective, a conclusion which Hemmings (1957) also reached as a result of investigations on the distribution of iodinated bovine and rabbit y-globulin following injection into the uterine lumen. There was no evidence of an intercellular route of transmission across the yolk-sac endoderm, although such a route has been suggested for the rat yolk sac on purely morphological grounds (Padykula, Deren & Wilson, 1966) and is implied by Kulangara & Schechtman (1962) in the suggestion that proteins reach the exocoel by molecular sieving across the yolk sac. However, an intercellular route of transmission to the exocoel is indicated, but across the paraplacental chorion. Here little intracellular uptake into the cytotrophoblast occurred, but there was observed with both techniques an intense staining of the basement membrane indicative of all proteins except human a-2 macroglobulin. Proteins appear to pass between cells in certain areas and reach the basement membrane directly. This route is supported by the localization of specific fluorescence around the cytotrophoblast and by electron microscope investigations on transmission of ferritin and imferon across the chorion, in which these electron-dense molecules have been seen in gaps leading to the basement membrane (A. E. Wild & B. S. Slade, in preparation). Larsen & Davies (1962) have also alluded to the chorion as a possible site for protein transfer, but on circumstantial evidence only. Davies (1959) also employing the fluorescent antibody technique, reported detecting bovine y-globulin in the yolk-sac exocoelomic mesothelium and interpreted this as indicating that protein had passed directly across the yolk sac. The same localization of proteins in the yolk-sac exocoelomic mesothelium was seen here, but it is clear that such protein is derived from the exocoelomic fluid, having traversed the chorion and not the yolk sac. Entry to the exocoel and subsequently the amniotic fluid by this route explains why there is a paucity of the large molecular weight proteins (Wild, 1965) and why there is no selection of y-globulins during transmission to these fluids (see Table 2 in this investigation; Batty et al. 1954; Kulangara & Schechtman, 1962) since the process clearly involves a selective ultrafiltration, as has been suggested for entry to the human amniotic fluid (Derrington & Soothill, 1961). It has been reasonably assumed (see Brambell, 1966) that the endodermal cell is the selective site during transmission of proteins to the rabbit foetal circulation. The results of the present investigation confirm this, for whilst all proteins 328 A. E. WILD investigated were similarly distributed in the endodermal cell, only rabbit and human y-globulin, and to a lesser extent human serum albumin, were detected in and below the basement membrane. Other possible sites for selection, such as the foetal reticulo-endothelial system and yolk-sac vascular mesenchyme and endothelium, can now be firmly ruled out. The failure to detect egg albumin in and below the basement membrane on those occasions when it was detected in the foetal blood, may be related to the low level of transmission of this protein across the yolk sac or to the existence of other sites of entry. Brambell, Hemmings & Morris (1964) have suggested that selective attachment of pinocytosed proteins to a finite number of receptors present on the limiting membrane of an absorptive vesicle, might provide protection from enzymic degradation after fusion with lysosomes. In some unknown way, after movement through the cell, protein is then presumed to be liberated from the vesicle into the intercellular space. Investigations on the ultrastructure of the yolk-sac endoderm (Petry & Kuhnel, 1965; Padykula et al. 1966; Slade, 1969) suggest that absorptive vesicles are formed as terminal dilations of the subapical canalicular system. This region gave a weak diffuse fluorescence in the present experiments, providing pictorial evidence of protein transport through the canalicular system and concentration in the underlying absorptive vesicles, since fluorescence here was much increased. From their later distribution in the endodermal cells it is clear that the vesicles are involved in transport of proteins towards the basement membrane, but whether or not all vesicles are involved is open to question. Proteins were associated with the perimeter of the vesicles or dispersed throughout them and this difference was not merely a function of the plane of the section. The characteristic breakages in the large, mainly supranuclear vesicles, may possibly be artifacts, but they feature prominently in electromicrographs of the yolk-sac endoderm. Padykula et al. (1966) observed a greater proportion of 'ruptured' vesicles in 21-day-old, compared to 14-day-old rat yolk-sac endoderm. These vesicles could therefore be part of the normal, intercellular degradation cycle, representing 'dead ends' as far as protein transport is concerned. Of the available, homologous y-globulin which is present in the yolk sac, only about 12% is transported to the foetal circulation (Hemmings, 1957) and it is possible that such protein is confined to the intact vesicles described. A necessary requisite for the hypothesis proposed by Brambell et al. (1964) is that more than one protein should be present in the same absorptive vesicle. In the double tracing experiment described, results were obtained which seem to confirm this. However, vesicles lying close to the basement membrane might have been expected to contain only human y-globulin if such protein was being protected, but this was not the case. Actual fusion of vesicles with the basement membrane was not readily observed, but it seems a more likely process for discharging proteins than liberation into the intercellular space. When such fusion does occur the vesicles may then contain only the transmitted protein. Subsequent passage of normal protein from the basement membrane to the lumen of Protein transmission 329 the vitelline vessel appears to be merely a process of diffusion through the loose vascular mesenchyme and the endothelia, but F.I.T.C.-conjugated human and rabbit y-globulin which reaches the vascular mesenchyme is in addition taken up into macrophages. Luse (1958) mentions detecting colloidal gold and fat in such cells. All conjugated proteins were also taken up into macrophages in the paraplacental chorion and into mesenchymal cells in the amnion. This anomalous treatment of conjugated proteins by macrophages has previously been reported by Nairn (1964) and is clearly related to the presence of the dye. Such cells probably have little role to play in the actual transport of proteins across the foetal membranes, but may further regulate their transmission by pinocytosing and digesting them. RESUME Transfer t de proteines a t ravers les membranes feet ales chez le lapin Le marquage des proteines par fluorescence, faisant intervenir des proteines conjuguees F.I.T.C. et la technique d'immuno fluorescence, a ete utilise pour etudier les sites et le mecanisme de transport de diverses proteines a travers les membranes fcetales du Lapin. II n'a pas ete possible de mettre en evidence un transfertintercellulaire de proteines a travers la splanchnopleure de la vesicule vitelline vers le ccelome extraembryonnaire, mais toutes les proteines se sont localisees dans des vesicules d'absorbtion de Pendoderme de la vesicule vitelline. Differentes proteines ne sont localisees de fa?on similaire dans les vesicules d'absorbtion dont la taille et I'aspect sont differents. Des vesicules eclatees caracteristiques ont ete observees et Ton a pu mettre en evidence la presence de plusieurs proteines dans chacune des vesicules absorbantes. Le role de Pendoderme de la vesicule vitelline en tant que site selectif pour le transfert des proteines vers la circulation foetale a ete confirme; seules les proteines rapidement decelees dans le serum foetal sont observees, a Pinterieur et au dessous de la membrane basale. II est demontre que le chorion paraplacentaire constitue bien le site de transfert des proteines vers le coelome extraembryonnaire, et que ce transfert s'effectue suivant un processus de diffusion. Contrairement aux proteines normales, les conjugues F.I.T.C. se localisent rapidement parmi les macrophages presents dans le chorion paraplacentaire et dans le mesenchyme vasculaire de la vesicule vitelline. Ces resultants sont discutes a la lumiere des travaux montrant que des differences existent entre le transfert de proteines vers les fluides fcetaux et vers le sang foetal et a la lumiere des hypotheses courantes rendant compte de la selection des proteines par Pendoderme vitellin. 1 am grateful to Mr M. Childes for his technical assistance and to the Science Research Council for their financial support. REFERENCES BATTY, I., BRAMBELL, F. W. R., HEMMINGS, W. A. & OAKLEY, C. L. (1954). Selection of anti- toxins by the foetal membranes of rabbits. Proc. R. Soc. B 142, 452-471. BRAMBELL, F. W. R. (1966). The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet ii, 1087-1093. BRAMBELL, F. W. R., HEMMINGS, W. A. & HENDERSON, M. (1951). Antibodies and Embryos. London: Athlone Press. BRAMBELL, F. W. R., HEMMINGS, W. A. & MORRIS, I. G. (1964). A theoretical model of y-globulin catabolism. Nature, Lond. 203, 1352-1355. 330 A. E. WILD DA VIES, J. (1959). In Transactions of the Vth Conference on Gestation, pp. 228-241. New York: Josiah Macy, Jr. Foundation. DERRINGTON, M. M. & SOOTHILL, J. F. (1961). An immunochemical study of the proteins of amniotic fluid and of maternal and foetal serum. /. Obstet. Gynaec. Br. Commonw. 68, 755-761. HEMMINGS, W. A. (1957). Protein selection in the yolk sac splanchnopleur of the rabbit: the total uptake estimated as loss from the uterus. Proc. R. Soc. B 148, 76-83. HEMMINGS, W. A. & BRAMBELL, F. W. R. (1961). Protein transfer across foetal membranes. Br. med. Bull. 17, 96-101. HEMMINGS, W. A. & JONES, R. E. (1962). The occurrence of macroglobulin antibodies in maternal and foetal sera of rabbits as determined by gradient centrifugation. Proc. R. Soc. B 157, 27-32. KAPLAN, K. C, CATSOULIS, E. A. & FRANKLIN, E. C. (1965). Maternal-foetal transfer of human immune globulins and fragments in rabbits. Immunology 8, 354-359. KULANGARA, A. C. & SCHECHTMAN, A. M. (1962). Passage of heterologous serum proteins from mother into foetal compartments in the rabbit. Am. J. Physiol. 203, 1071-1080. LARSEN, J. F. & DAVIES, J. (1962). The paraplacental chorion and accessory foetal membranes of the rabbit. Histology and electron microscopy. Anat. Rec. 143, 27-45. LUSE, S. A. (1958). Morphologic manifestations of uptake of materials by the yolk sac of the pregnant rabbit. In Transactions of the IVth Conference on Gestation. New York: Josiah Macy, Jr. Foundation. NAIRN, R. C. (1964). Fluorescent Protein Tracing, pp. 22, 90. London: Livingstone. PADYKULA, H. A., DEREN, J. J. & WILSON, H. T. (1966). Development of structure and function in the mammalian yolk sac. 1. Development, morphology and vitamin B12 uptake of the rat yolk sac. Devi Biol. 13, 311-348. PETRY, G. & KUHNEL, W. (1965). Der Feinbau des Dottersackepithels und Dessen Beziehung zur Eiweissresorption (Kaninchen). Z. Zellforsch. mikrosk. Anat. 65, 27-46. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. /. Histochem. Cytochem. 10, 250-256. SLADE, B. S. (1969). Studies on Protein Transmission across the Rabbit Yolk Sac Splanchnopleur. Ph.D. Thesis, University of Southampton. WILD, A. E. (1965). Protein composition of the rabbit foetal fluids. Proc. R. Soc. B 163, 90-115. WILD, A. E. (1966). Protein transmission across the placenta and foetal membranes. Sci. Prog., Oxford 54, 351-365. (Manuscript received 3 December 1969)