/ . Embryol. exp. Morph. Vol 24, 2, pp. 313-330, 1970 3 1 3

Printed in Great Britain

Protein transmission across therabbit foetal membranes

By A. E. WILD1

From the Department of Zoology, University of Southampton

SUMMARY

Fluorescent protein tracing, involving F.I.T.C.-conjugated proteins and the fluorescentantibody technique, was employed to study the sites and mechanism of transport of a varietyof proteins across the rabbit foetal membranes.

No evidence was found for an intercellular transmission of proteins across the yolk sacsplanchnopleure to the exocoel, but all proteins were shown to become localized in absorptivevesicles in the yolk-sac endoderm.

Different proteins became similarly localized in absorptive vesicles having different sizesand profiles. Characteristic broken vesicles were present, and more than one protein wasdemonstrated in each absorptive vesicle.

The yolk-sac endoderm was confirmed as the selective site for transmission of proteins tothe 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 theexocoel and the process to be one of diffusion.

Unlike normal proteins, F.l.T.C. conjugates readily became localized within macrophagespresent in the paraplacental chorion and yolk-sac vascular mesenchyme.

These findings are discussed in the light of differences previously shown to occur betweentransmission of proteins to the foetal fluids and to the foetal blood, and in the light of acurrent 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 theexocoelomic, amniotic and allantoic fluids. A greater proportion of albuminwas also found in these fluids when comparison was made with the foetal andmaternal sera. These findings pointed to a selective transmission of proteinsbased on molecular size and similar findings for the amniotic fluid of otherspecies are consistent with this view (for details see review by Wild, 1966). Incontrast, transmission of proteins to the foetal rabbit circulation is clearlyindependent 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 transmittedselectively to the foetal circulation but non-selectively to the fluids (Batty,Brambell, Hemmings & Oakley, 1954; Kulangara & Schechtman, 1962). Whilstno doubt exists that the yolk-sac splanchnopleure is the site for transmission ofproteins to the foetal circulation (Brambell, Hemmings & Henderson, 1951) thesite for transmission to the exocoel has been a matter for more speculation.Kulangara & Schechtman (1962) suggested that proteins reached the exocoel bydiffusion 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 emphasizedits possible role since in in vitro dialysis experiments it was much more permeableto protein than the yolk sac (Wild, 1965).

In the present investigation use has been made of fluorescent protein tracingin order to locate the site and elucidate the mechanism of transmission to theexocoel in vivo, and also to throw light on the selective mechanism involved intransfer 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), boviney-globulins (Armour, Fraction 11), human serum albumin (Mann Laboratories),and egg albumin (Armour) was carried out according to Nairn (1964) exceptthat 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. wascontinued overnight at 4 °C. Non-reacted F.I.T.C. was removed by SephadexG-25 chromatography and the conjugates brought back to their originalvolumes by ultrafiltration in Visking tubing. Final concentrations of albuminswere 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, alsoto 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 labelledanti-human serum albumin were obtained commercially (Mann Laboratories).Immunoelectrophoresis employing the conjugated absorbed antisera revealedonly 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 afterlaparotomy injected into the uterine lumen of Dutch rabbits, 26-28 dayspregnant, at a dose of 1 ml/conceptus. At various time intervals, ranging from15 min to 8 h, rabbits were killed with nembutal and the conceptuses exposed.After washing with normal saline, areas of yolk sac, paraplacental chorion andamnion 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, exocoelomicand amniotic fluids and foetal blood were collected and tested for the presenceof heterologous proteins by means of the interfacial ring test and in some casesimmunoelectrophoresis. Subsequent dehydration, clearing and embedding ofthe membranes was carried out according to Nairn (1964). After dewaxing inthree changes of xylene, sections (4 /t) were passed through three changes of100% 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/onepart phosphate-buffered saline. Membranes from conceptuses of non-injectedhorns, similarly processed, were used as controls. Sections were examined foru.v. fluorescence in a Wild M 20 microscope. Photographs were taken in colourusing Kodak High Speed Ektachrome and in black and white using Tri-X.

For tracing by the fluorescent antibody technique, normal human serum wasused as a source of human y-globulin, a-2 macroglobulin and albumin, andwas supplemented with egg albumin at a concentration of 30 mg/ml. Solutionscontaining 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 accordingto the technique described by Sainte-Marie (1962). In order to reduce non-specific staining, conjugated antisera were absorbed first with pig liver powder(Burroughs Wellcome) followed by one or sometimes two absorptions withfoetal membrane powder prepared from chorion, yolk sac and amnion ofconceptuses not exposed to heterologous proteins. Rabbit antibovine y-globulinserum was found to cross-react slightly with human y-globulin, and to removesuch cross-reacting antibodies, 5 ml batches of conjugated antiserum wereabsorbed with 50 mg human y-globulin for 24 h at 4 °C. The precipitate wasthen removed by filtration. Absorbed, specific conjugated antisera were appliedto dewaxed sections for 30 min in a saturated environment. Sections were thenwashed for 1 h in two changes of phosphate-buffered saline. Blocking tests werecarried out with non-conjugated antisera but the best controls were sections ofmembranes 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 localizedin vesicles in the endodermal cells of the yolk-sac splanchnopleure (Figs. 1, 2).There was no indication that conjugates passed through, or were dischargedinto, 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 appear-ance to control tissue. In such cases the conjugates had presumably failed tospread around the conceptus, or had done so unevenly.

316 A. E. WILD

A

VHS

Protein transmission 317

The vesicles showed a variation in size and in distribution and intensity offluorescence. After 15 and 30min exposure to conjugates, the vesicles weremainly sub-apically located (Fig. 1A, B) and then became more randomlydistributed (Fig. 1C-E; Fig. 2A, C). Fluorescence was distributed throughoutvesicles, or it was confined to the perimeter, or unevenly distributed on theperimeter of intact and broken vesicles (Fig. 1B, E; Fig. 2 A, C). Broken vesicleswere the largest and usually confined to the supra-nuclear region, whilst smallervesicles were more randomly distributed.

Bovine, human, and rabbit y-globulin, human serum and egg albumin con-jugates, all showed a similar localization of fluorescence in vesicles which weresimilarly distributed within the endodermal cell. However, only with human andrabbit y-globulin conjugates was fluorescence ever detected in the basementmembrane of the yolk-sac endoderm, in the vascular mesenchyme, or in thelumen 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 = canali-cular system; cyt = cytotrophoblast; e = erythrocyte; em = exocoelomic meso-thelium; 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-globulinF.l.T.C. conjugate for 30 min. The conjugate is confined to vesicles situated mainlyin the apical regions of the yolk-sac endoderm. x 100. Bright-field fluorescence1-̂ min exposure.

(B) Another area of yolk-sac splanchnopleure of rabbit no. 20 at higher magnifica-tion. The conjugate is confined to intact and broken vesicles. Erythrocytes in avitelline vessel show autofluorescence. x 500. Dark-field fluorescence, l^minexposure.

(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 endo-dermal cells. The conjugate is absent from the basement membrane but occurs in ashort region of the exocoelomic mesothelium (arrowed) indicating derivation fromthe exocoel. x 100. Bright-field fluorescence, \\ min exposure.

(D) Yolk-sac splanchnopleure of rabbit no. 48 exposed to human y-globulinF.l.T.C. conjugate for 2^h. Note the presence of the conjugate in the basementmembrane and in macrophages in the vascular mesenchyme. x 100. Bright-fieldfluorescence, 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. Thelargest are broken and fluorescence is less intense in these. A vesicle (arrowed)appears to lie within the basement membrane, which shows a diffuse distribution ofthe conjugate. Other vesicles lie in close apposition to the basement membrane.Macrophages containing small, intensely fluorescent vesicles lie close to vitellinevessels. Specific fluorescence indicative of the conjugate is present along the exo-coelomic 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 mem-brane and on rare occasions apparently within it (Fig. 1E), but whilst fusion ofvesicles with, or discharge of their contents into the basement membrane seemsthe only way of accounting for the observed specific fluorescence, more con-vincing evidence of such a process was not observed. Diffuse specific fluorescencewas also evident in the vascular mesenchyme and in the endothelium of thevitelline vessels, and where red blood cells remained in the vessel lumen, it wasfrequently seen around them (Fig. 2 A). A further striking feature with rabbitand human y-globulin conjugates was their localization in macrophages whichfrequently bordered the vitelline vessels (Fig. ID, E) or lay in the vascularmesenchyme in close apposition to the basement membrane (Fig. 2A, B).Within the macrophages fluorescence was confined to much smaller vesiclesthan observed in the endodermal cells. All conjugates were unevenly distributedalong the exocoelomic mesothelium (see Fig. 1C, E) and at least in the case ofbovine y-globulin, human and egg albumin, this represented a derivation fromthe 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. Amacrophage lies in close contact with the basement membrane. Diffuse fluorescenceis present along the brush border of the yolk-sac endoderm. x 500. Dark-fieldfluorescence, \% 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 base-ment membrane and underlying macrophages show presence of the conjugate,which is also dispersed through the vascular mesenchyme. x 400. Bright-field fluo-rescence, 1-̂ min exposure.(C) An area of yolk-sac splanchnopleure of rabbit no. 48 in which broken vesiclesare particularly prominent but conjugate is absent from the basement membraneand vascular mesenchyme. Note the variation in intensity of fluorescence within thevesicles. 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, brightlyfluorescing droplets of conjugate, can be seen in the loose mesenchymal tissue. Thebasement membrane of the cytotrophoblast and the mesenchymal tissue itself showmuch 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 thecytotrophoblast and dispersed throughout the loose mesenchymal tissue and exo-coelomic mesothelium. Specific fluorescence also occurs around the cytotrophoblastcells in some areas, but not within them, x 100. Bright-field fluorescence, H minexposure.(F) Paraplacental chorion of rabbit no. 34 exposed to egg albumin F.I.T.C. conju-gate for 3 h. In this field the conjugate is heavily localized in the cellular debris andsurrounds giant cells in an area of giant-cell proliferation. There has been little pene-tration beyond the cytotrophoblast basement membrane, x 100. Bright-fieldfluorescence, H min exposure.

320 A. E. WILD

Protein transmission 321albumin from in and below the basement membrane was generally correlatedwith negative precipitin tests for these proteins in the foetal blood, but eggalbumin was sometimes detected in low titre when there was no evidencefor its transmission to the vitelline vessels. However, all conjugates werereadily detected in the exocoelomic fluid and within 15min of injection inthe case of that conceptus nearest the injection site. Entry into the exocoelwas clearly by way of the paraplacental chorion, as evidenced by diffusefluorescence around the trophoblastic giant cells and cytotrophoblast, and byintense fluorescence of the basement membrane of the cytotrophoblast and ofthe loose mesenchymal tissue and exocoelomic mesothelium (Fig. 2E, F). Allconjugates, again in the form of small vesicles, were found in macrophagesdispersed through the mesenchymal tissue (Fig. 2D). Such macrophages variedconsiderably in density throughout the chorion, but they were more abundantin longer term experiments and easily observed in living tissue spread as a thinsheet and exposed to u.v. light (Fig. 3 A). Diffuse specific fluorescence was alsoobserved in the extracellular ground substance and on the myofibrils present inthe amnion. Again in longer term experiments (after 2 h), all conjugates wereseen 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 whenspecific fluorescence was absent from the basement membrane and vitelline

Fig. 3. (A) Spread of living chorion from rabbit no. 34 showing an area rich inmacrophages in which the conjugate has become localized. Focused through theexocoelomic 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 humanserum supplemented with egg albumin, and treated with rabbit antihuman y-globulinF.l.T.C.-conjugated antiserum. Specific fluorescence indicative of human y-globulinis confined to the brush border and subapical vesicles in the endoderm. Fluorescenceis also present along the exocoelomic mesothelium indicating protein derived fromthe exocoel. x 100. Bright-field fluorescence, \% min exposure.

(D) Yolk-sac splanchnopleure of rabbit no. 40 treated with rabbit anti-egg albuminF.l.T.C.-conjugated antiserum. Weak diffuse specific fluorescence indicative ofprotein is present in the subapical canalicular system and concentrated below thisin terminal absorptive vesicles, x 500. Dark-field fluorescence, \\ min exposure.(E) Yolk-sac splanchnopleure of rabbit no. 40 treated with rabbit antihumany-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 fluo-rescence, \\ min exposure.(F) Yolk-sac splanchnopleure of rabbit no. 39 exposed for 30 min to normalhuman serum supplemented with egg albumin, and treated with rabbit antihumany-globulin F.l.T.C.-conjugated antiserum. Protein is present in intact and brokenvesicles, and on the surface of erythrocytes (arrowed). The basement membraneshows no specific fluorescence although vesicles lie in close apposition to it. x 500.Dark-field fluorescence, \\ min exposure.

322 A. E. WILD

Protein transmission 323

vessels, the possibility that protein and fluorescein became dissociated as a resultof 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 inphosphate-buffered saline, and the foetal membranes examined after 3 hexposure, fluorescence was again detected in vesicles in the yolk-sac endoderm.These vesicles were much smaller than those observed with the conjugates andthere was no fluorescence in or below the basement membrane. In the chorionbright diffuse fluorescence was distributed throughout the tissue, but no macro-phages were located. Sections of yolk sac exposed to egg albumin conjugateswere also treated with rabbit anti-egg albumin F.I.T.C. This treatment failedto reveal any further localization of fluorescence, either in or below the base-ment membrane, or in the vitelline vessels. In order to check whether thefluorescence observed in the basement membrane, the vascular mesenchyme andmacrophages, was in fact human y-globulin conjugate and not free fluorescein,sections of yolk sac exposed to human y-globulin conjugate for 2\ h weretreated with rabbit antihuman y-globulin R.B. 200 Cl. This resulted in ayellowish fluorescence in previously apple-green fluorescing sites and as opposedto 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 andy-globulin; bovine y-globulin and egg albumin) were again detected withinvesicles in the yolk-sac endoderm, as evidenced by specific fluorescence aftertreatment of sections with specific conjugated antisera (see Figs. 3-5). As withthe direct tracing technique, only a non-selective intracellular route of entry wasindicated. This was well illustrated in experiments of 15min duration, afterwhich 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 normalhuman serum supplemented with egg albumin and treated with rabbit anti-eggalbumin F.I.T.C.-conjugated antiserum. Note the variation in distribution andintensity of fluorescence within the vesicles. Many lie close to the basement mem-brane which shows no specific fluorescence, x 500. Dark-field fluorescence, 1$ minexposure.(B) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihumany-globulin F.I.T.C.-conjugated antiserum. Note that the vesicles show a similarvariation in intensity and distribution of fluorescence when compared with those inFig. 4 (A), indicating a similar distribution of the two proteins in the endoderm.The basement membrane, vascular mesenchyme and endothelium all show intensespecific fluorescence, x 400. Bright-field fluorescence, 1̂ - min exposure.

(C) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihumany-globulin F.I.T.C.-conjugated antiserum. In this field broken vesicles are moreevident and intense specific fluorescence indicative of human y-globulin is presentwithin a vessel. The basement membrane shows no specific fluorescence in thisregion, x 500. Dark-field fluorescence, 1 | min exposure.21 E M B 2 4

324 A. E. WILD

and concentrated immediately below this into vesicles (Fig. 3C-E). Greaternon-specific fluorescence was observed with this technique, especially in nucleiand in erythrocytes, but specific fluorescence within vesicles was never observedin treated control tissue and blocking tests carried out with non-conjugatedantisera considerably reduced specific fluorescence in experimental material.Distribution of fluorescence within vesicles was similar to that encountered withthe 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 ofmembranes to antigen. Human serum albumin could also be detected in thesesites, but only after longer exposure and with much less intensity of specificfluorescence. Although vesicles containing egg albumin and bovine y-globulinlay close to the basement membrane (Fig. 4A; Fig. 5B, D) they were neverdetected 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 macro-phages, either in the vascular mesenchyme or in the paraplacental chorion, andthis provided the only striking difference between treatment of conjugated andnormal proteins. Bovine y-globulin was sometimes observed in macrophages inthe paraplacental chorion (Fig. 5D) and was present as droplets in mesenchymalcells of the amnion. After 15 min exposure, proteins were again detected in theexocoelomic fluid when they were absent from the foetal serum. Results illus-trating this in a typical experiment are shown in Table 1. This rapid entry ofproteins to the exocoel was again correlated with the presence of specific fluo-rescence indicative of all proteins (except human a-2 macroglobulin) in andaround multinucleate giant cells, around the cytotrophoblast and in its basementmembrane, and dispersed through the loose mesenchymal tissue and exocoelomicmesothelium (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 normalhuman serum supplemented with egg albumin and treated with rabbit antihumany-globulin F.I.T.C.-conjugated antiserum. Specific fluorescence is present in debrissurrounding giant cells, in the cytotrophoblast basement membrane and dispersedthroughout the loose mesenchymal tissue. Nuclei of cytotrophoblast and giant cellsshow non-specific fluorescence (arrow), x 100. Bright-field fluorescence, l^minexposure.

(B) Yolk-sac splanchnopleure of rabbit no. 37 exposed for 5 h to normal humanserum supplemented with egg albumin and treated with rabbit anti-egg albuminF.i.T.C.-conjugated antiserum. Specific fluorescence is confined to vesicles in theyolk-sac endoderm. Nuclei in the vascular mesenchyme (arrowed) show non-specificfluorescence, x 100. Bright-field fluorescence, 1̂ min exposure.(C) Yolk-sac splanchnopleure of rabbit no. 37 treated with rabbit antihumany-globulin F.T.T.C.-conjugated antiserum. Note intense specific fluorescence in thevascular mesenchyme and lumen of vitelline vessel, x 100. Bright-field fluorescence,\\ min exposure.(D) Yolk-sac splanchnopleure and paraplacental chorion of rabbit no. 51 exposedfor 3 h to a mixture of human and bovine y-globulins and treated with rabbit anti-bovine y-globulin F.i.T.C.-conjugated antiserum. Specific fluorescence is confinedto vesicles in the yolk-sac endoderm and in the chorion to the loose mesenchymaltissue and macrophages. x 100. Bright-field fluorescence, 1^ min exposure.(E) Yolk-sac splanchnopleure of rabbit no. 51 treated with rabbit antihumany-globulin F.i.T.C.-conjugated antiserum. Note intense specific fluorescence in thebasement 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 thenon-selective entry of these proteins to the fluids. Results for a typical experi-ment are shown in Table 2. Specific fluorescence representing protein derivedfrom the exocoel was again evident in intermittent areas along the exocoelomicmesothelium of the yolk sac (Fig. 3C).

Table 1. Transmission of human y-globulin and egg albumin to the fluids and bloodof the single conceptus of rabbit no. 40,15 min after exposing it to 1 ml of normalhuman serum supplemented with 3% egg albumin

Foetal fluid

ExocoelomicAmnioticSerumInjected serum

Titre againstrabbit

anti-eggalbumin

12832

— ve1048000

Titre againstrabbit

anti-humany-globulin

8— ve— ve

32000

Table 2. Transmission of human and bovine y-globulin to the fluids of the firstconceptus of rabbit no. 51, 3 h after exposing it to a mixture of both proteins

Foetal fluid

ExocoelomicAmnioticSerumInjected solution

Titre againstrabbit

anti-boviney-globulin

6432

— ve8000

Titre againstrabbit

anti-humany-globulin

643232

8000

In order to determine whether or not human y-globulin and egg albumin werelocalized in the same vesicles, sections of yolk sac exposed to human serum sup-plemented with egg albumin were treated first with F.I.T.C.-labelled antiserumto egg albumin and the fluorescence of the same fields compared after subse-quent treatment with R.B. 200 Cl-labelled antiserum to human y-globulin.Vesicles previously fluorescing apple green, gave an intermediate orange-yellowfluorescence after such treatment, and no fields were found in which apple-greenfluorescing vesicles co-existed with reddish orange vesicles. Such results wereinterpreted as indicating that all vesicles contained both proteins.

DISCUSSION

The use of F.I.T.C. conjugates for tracing proteins presents certain limitationsnot encountered with the fluorescent antibody technique. The fluorochromemay 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 fluorescentantibody technique, on the other hand, presents problems not encountered withF.l.T.C. conjugates, namely possible loss of antigenicity and non-specificstaining. Thus findings common to both techniques are better guides to themechanisms involved in uptake and transport of proteins than are findingsattributable 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 givesa visual demonstration that protein uptake is non-selective, a conclusion whichHemmings (1957) also reached as a result of investigations on the distributionof iodinated bovine and rabbit y-globulin following injection into the uterinelumen. There was no evidence of an intercellular route of transmission acrossthe yolk-sac endoderm, although such a route has been suggested for the ratyolk sac on purely morphological grounds (Padykula, Deren & Wilson, 1966)and is implied by Kulangara & Schechtman (1962) in the suggestion thatproteins reach the exocoel by molecular sieving across the yolk sac. However, anintercellular route of transmission to the exocoel is indicated, but across theparaplacental chorion. Here little intracellular uptake into the cytotrophoblastoccurred, but there was observed with both techniques an intense staining of thebasement membrane indicative of all proteins except human a-2 macroglobulin.Proteins appear to pass between cells in certain areas and reach the basementmembrane directly. This route is supported by the localization of specific fluo-rescence around the cytotrophoblast and by electron microscope investigationson transmission of ferritin and imferon across the chorion, in which theseelectron-dense molecules have been seen in gaps leading to the basement mem-brane (A. E. Wild & B. S. Slade, in preparation). Larsen & Davies (1962) havealso alluded to the chorion as a possible site for protein transfer, but on circum-stantial evidence only.

Davies (1959) also employing the fluorescent antibody technique, reporteddetecting bovine y-globulin in the yolk-sac exocoelomic mesothelium and inter-preted this as indicating that protein had passed directly across the yolk sac.The same localization of proteins in the yolk-sac exocoelomic mesothelium wasseen 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 andsubsequently the amniotic fluid by this route explains why there is a paucity ofthe large molecular weight proteins (Wild, 1965) and why there is no selectionof y-globulins during transmission to these fluids (see Table 2 in this investiga-tion; Batty et al. 1954; Kulangara & Schechtman, 1962) since the processclearly involves a selective ultrafiltration, as has been suggested for entry to thehuman amniotic fluid (Derrington & Soothill, 1961).

It has been reasonably assumed (see Brambell, 1966) that the endodermalcell is the selective site during transmission of proteins to the rabbit foetal circu-lation. 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 andhuman y-globulin, and to a lesser extent human serum albumin, were detectedin and below the basement membrane. Other possible sites for selection, suchas the foetal reticulo-endothelial system and yolk-sac vascular mesenchyme andendothelium, can now be firmly ruled out. The failure to detect egg albumin inand below the basement membrane on those occasions when it was detected inthe foetal blood, may be related to the low level of transmission of this proteinacross the yolk sac or to the existence of other sites of entry. Brambell,Hemmings & Morris (1964) have suggested that selective attachment of pino-cytosed proteins to a finite number of receptors present on the limiting membraneof an absorptive vesicle, might provide protection from enzymic degradationafter fusion with lysosomes. In some unknown way, after movement through thecell, protein is then presumed to be liberated from the vesicle into the intercellularspace. Investigations on the ultrastructure of the yolk-sac endoderm (Petry &Kuhnel, 1965; Padykula et al. 1966; Slade, 1969) suggest that absorptivevesicles are formed as terminal dilations of the subapical canalicular system.This region gave a weak diffuse fluorescence in the present experiments, pro-viding pictorial evidence of protein transport through the canalicular systemand concentration in the underlying absorptive vesicles, since fluorescence herewas much increased. From their later distribution in the endodermal cells it isclear that the vesicles are involved in transport of proteins towards the base-ment membrane, but whether or not all vesicles are involved is open to question.Proteins were associated with the perimeter of the vesicles or dispersed through-out them and this difference was not merely a function of the plane of thesection. The characteristic breakages in the large, mainly supranuclear vesicles,may possibly be artifacts, but they feature prominently in electromicrographsof the yolk-sac endoderm. Padykula et al. (1966) observed a greater proportionof 'ruptured' vesicles in 21-day-old, compared to 14-day-old rat yolk-sac endo-derm. These vesicles could therefore be part of the normal, intercellular degrada-tion cycle, representing 'dead ends' as far as protein transport is concerned. Ofthe available, homologous y-globulin which is present in the yolk sac, onlyabout 12% is transported to the foetal circulation (Hemmings, 1957) and it ispossible 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 seemto confirm this. However, vesicles lying close to the basement membrane mighthave been expected to contain only human y-globulin if such protein was beingprotected, but this was not the case. Actual fusion of vesicles with the basementmembrane was not readily observed, but it seems a more likely process for dis-charging proteins than liberation into the intercellular space. When such fusiondoes occur the vesicles may then contain only the transmitted protein. Subse-quent 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 loosevascular mesenchyme and the endothelia, but F.I.T.C.-conjugated human andrabbit y-globulin which reaches the vascular mesenchyme is in addition takenup into macrophages. Luse (1958) mentions detecting colloidal gold and fat insuch cells. All conjugated proteins were also taken up into macrophages in theparaplacental chorion and into mesenchymal cells in the amnion. This anomaloustreatment of conjugated proteins by macrophages has previously been reportedby Nairn (1964) and is clearly related to the presence of the dye. Such cellsprobably have little role to play in the actual transport of proteins across thefoetal membranes, but may further regulate their transmission by pinocytosingand 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 conjugueesF.I.T.C. et la technique d'immuno fluorescence, a ete utilise pour etudier les sites et lemecanisme 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 traversla splanchnopleure de la vesicule vitelline vers le ccelome extraembryonnaire, mais toutes lesproteines se sont localisees dans des vesicules d'absorbtion de Pendoderme de la vesiculevitelline.

Differentes proteines ne sont localisees de fa?on similaire dans les vesicules d'absorbtiondont la taille et I'aspect sont differents. Des vesicules eclatees caracteristiques ont ete observeeset Ton a pu mettre en evidence la presence de plusieurs proteines dans chacune des vesiculesabsorbantes.

Le role de Pendoderme de la vesicule vitelline en tant que site selectif pour le transfert desproteines vers la circulation foetale a ete confirme; seules les proteines rapidement deceleesdans 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 desproteines vers le coelome extraembryonnaire, et que ce transfert s'effectue suivant un processusde diffusion.

Contrairement aux proteines normales, les conjugues F.I.T.C. se localisent rapidementparmi les macrophages presents dans le chorion paraplacentaire et dans le mesenchymevasculaire de la vesicule vitelline.

Ces resultants sont discutes a la lumiere des travaux montrant que des differences existententre le transfert de proteines vers les fluides fcetaux et vers le sang foetal et a la lumiere deshypotheses 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 ResearchCouncil for their financial support.

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(Manuscript received 3 December 1969)