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  y.  Cell Sci.  25, 293-312 (1977)  293 Printed in Great Britain DIFFERENTIATED REGIONS OF HUMANPLACENTAL CELL SURFACE ASSOCIATEDWITH EXCHANGE OF MATERIALS BETWEENMATERNAL AND FOETAL BLOOD:COATED VESICLES C. D.OCKLEFORD  AND  A. WHYTE Department  of  Pathology,  The  University  of  Cambridge, Tennis Court  Road Cambridge  CBz  1QP, England SUMMARYCoated vesicles may be an important component  of  the micropinocytic system  of  the humanplacenta. Regions  of  very dense reaction with glycocalyx stains  are  restricted  to  membraneswithin forming  and  fully formed coated vesicles. This  is  interpreted  as  evidence againstpermanently grouped specific binding sites having  a  role  in  the selective uptake of materials  by micropinocytosis,  and as  support  for  theories  of  coated-vesicle formation which take intoaccount  the  dynamic nature  of  membrane components.  The  pyroantimonate precipitationtechnique which was employed  in an  attempt  to  localize cations  in  placental tissue  at  termresulted in the deposition of electron-dense material in coated vesicles and basement membrane.Examination  of  the distribution  of  coated vesicles  in  placental tissue explants  at  8-13 weeksof gestation revealed  a  restricted distribution  of  these organelles. Probably more than 89 of coated vesicles lie within the largest vesicles' diameter from the cell surface.Placental coated vesicles were isolated and examined using negative staining. A polygonallypatterned structure was apparent on their surfaces. Analysis  of  the isolated fraction  of  coatedvesicles using sodium dodecyl sulphate polyacrylamide gel electrophoresis shows the presenceof a major protein of molecular weight 180000. This is the same molecular weight that has beengiven  for  clathrin,  the  major protein  of the  raised polygonally patterned structure  on the cytoplasmic surface  of  coated vesicles from other sources.INTRODUCTION Much is known of the physiology of transport of materials into the human placenta(Miller & Berndt, 1975) but there is a lack of data on the organization of the dynamicstructures involved in uptake. Two major questions arise from the physiological work.Firstly, the processes  of  uptake are unexplained. Secondly, the placenta  is  able  to collect certain materials from their relatively dilute state in maternal blood and main-tain them  at a  higher concentration  in  the foetal circulation. Materials taken up andconcentrated  in  this way include proteins, polysaccharides, fats, certain vitamins andinorganic ions (Brambell, 1970; Llewellyn-Jones, 1969).Allison & Davies (1974) have gathered a consensus of support  for  their view thatendocytic organelles are separable by their different structural natures. These differ-ences in structure may correlate with the physiologically distinct classes  of  endocyticactivity. For example, there are drugs which differentially suppress certain classes of  294  C. D.  Ockleford  and A. Whyte activity (Wills, Davies, Allison & Haswell, 1972). In addition, different forms ofendocytosis have differing energy requirements (Simpson & Spicer, 1973). Micro-pinocytosis by coated vesicle formation represents an area where understanding of theunderlying motility is developing (Kanaseki & Kadota, 1969; Ockleford, 1976).One approach to the problems of the motile processes of uptake in the placenta andthe phenomenon of selection is to study particular endocytic organelles. Coatedvesicles are a common constituent of cells and are found in the placenta. They occurwith great abundance in cells which selectively absorb protein (Lloyd & Beck, 1974).Since the human placenta takes up proteins selectively, there is a comparative cellularanatomical argument for making coated vesicles the organelles of choice in this study.This research has been undertaken at a favourable moment in the developmentof techniques for the study of these organelles. Pearse (1975) has recently developeda method for the isolation of porcine brain coated vesicles. This has proved adaptableto the placenta and has enabled us to complement our fine-structural data fromsectioned material with structural data from negatively stained, isolated vesicles andwith data from polyacrylamide gel electrophoresis. Using this range of techniques wehave sought information that could be used to gain an understanding of the dynamicevents involved in selective uptake by coated-vesicle formation in the transportprocesses which support foetal development. MATERIALS AND METHODS Trophoblast tissue Portions of human trophoblast tissue were recovered shortly after therapeutic terminationsof pregnancy performed in the  8-12th  week. Groups of about 20 chorionic villi were isolatedby dissection and washed  3  times in Hanks' Balanced Salts Solution (BSS) (Flow Laboratories,Irvine, Scotland). Unless stated specifically to the contrary, this was the tissue used. Termtrophoblast was obtained  post-partum  and was treated similarly. Trophoblast cells werecultured as described by Loke & Borland (1970). Transmission election microscopy The methods routinely used for fixation and embedding and for ultrathin sectioning aredescribed fully in a previous publication (Ockleford, 1975).Samples containing coated vesicles were negatively stained using  1  uranyl acetate (Huxley,1963). They were supported on carbon-coated copper grids.Chorionic villi were stained with ruthenium red by the method of Luft (1965); by adding tothe tissue a mixture of 1-8 ml of  10  ruthenium red together with 23 ml of a mixture of thesolutions A and B described earlier (Ockleford, 1975); and by preincubating the tissue for10 min in i-8 ml of 10 ruthenium red and 23 ml of Hanks' BSS prior to fixation.Chorionic villi were stained with Alcian blue by using a  1  solution of the dye (Behnke &Zelander, 1970; Rothman, 1970).The tissue was stained with colloidal iron using the method first used for isolated membraneby Marx, Graf   Wesemann (1973).  Vibrio comma cholerae)  neuraminidase (Behringwerke A.G.Marburg Lahn) at activities of 50 and 500 U/ml was used to predigest one group of tissuesamples. Of these, some samples were esterified using  CVIN  HC1 dissolved in methanol andothers were esterified and later saponified using a  1  solution of KOH in 80 ethanol asthese authors have recommended (Marx  et al.  1973).For tannic acid fixation tissue-cultured trophoblast cells were fixed using  8  tannic acid and  Coated vesicles of human placenta  295 a 1 solution of the penetration agent digitonin (Tilney  et al.  1973). The cells were embeddedusing Spurr's (1969) low viscosity resin.Cation precipitation was accomplished using the method of Tandler, Libanati & Sanchis(1970). To increase the insolubility of the cationic precipitate the tissue was heated for 5 minat 95  C C in a half-saturated solution of potassium pyroantimonate and then rinsed twice in ice-cold distilled water as these authors recommended. Term placental tissue was used for cationprecipitation. Distribution of coated vesicles The surface of the syncytiotrophoblast where caveolae occur is highly convoluted. There-fore, it is not obvious whether some membrane-bounded electron-lucent areas represent truevacuoles within the cell or transverse sections through invaginating membrane pockets whichare still continuous with the cell surface. This complicates an analysis of the distribution ofcoated vesicles based upon measured distance from the cell surface. Consequently the data havebeen treated in 2 ways. Firstly, measurements have been made of the distance of coatedvesicles from the nearest definitely recognizable portion of the cell surface. This type of treat-ment gives an overestimate of the real average distance from the cell surface but gives a firmupper limit to the value. The second more uncertain method used any available indicator (forexample faint traces of contents or vesicle shape and size) to designate some ' apparent vesicles'as sections through invaginations of the cell surface membrane. Then, if coated vesicle profileswere closer to these than they were to the definitely recognizable cell surface, this new lowervalue was substituted in an attempt to achieve a more accurate estimate of the average distancefrom the cell surface. Only sections that were approximately perpendicular to the long axis ofthe villi were used. Any variation from the vertical in the section plane would be likely toincrease the apparent average distance of coated vesicles from the cell surface obtained usingeither method. Both potential errors lead to overestimation of the average distance of coatedvesicles from the cell surface.A comparison of distribution and number of coated vesicles in cultured trophoblast cellswith and without 0-02 ethylenediamine tetra-acetic acid (EDTA) added to the culturemedium was undertaken. The cells were incubated in the presence of EDTA at 37 °C for15 min; they were removed from the culture flasks by shaking or by means of a rubber police-man, fixed as described earlier and embedded in low-viscosity resin (Spurr, 1969). Isolation of coated vesicles Coated vesicles were isolated using the technique devised by Pearse (1975) with the followingmodifications. Tissue was disrupted (3 x 5-s periods) using an MSE homogenizer operated athalf speed. Samples were examined electron microscopically at all stages of purification.Coated vesicles were collected after the first sucrose density gradient step (5-60 ) for sub-sequent examination by electron microscopy and polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis Electrophoresis of the proteins of the coated vesicle fraction was undertaken using 7-5 polyacrylamide gels without stacking gels (Laemmli, 1970). Runs were initiated at  1  mA per gel.Once the dye front had migrated 1 cm into the gel the current was increased to 2 mA per gel.Gels were fixed for 30 min using 10 glacial acetic acid in 50 aqueous methanol. Protein-containing bands were stained for 30 min using a 1-25 solution of Coomassie brilliant blue inthe same solvent. Gels were destained at 60 °C using several changes of 7 acetic acid in 10 aqueous methanol. Mobilities of the major protein bands were expressed according to theformula of Weber & Osborn (1969). Molecular weights of proteins from the coated-vesiclefraction were assessed from a calibration graph. This was plotted using the mobilities of proteinsof known molecular weight determined from gels run simultaneously with those containingcoated-vesicle protein. The proteins used for calibration were pepsin (Sigma), mol. wt, 35000;bovine serum albumin fraction V (Sigma), mol. wt, 68000; and /?-galactosidase (Sigma grade m),  mol. wt of oligomer, 130000. Gels were scanned using a Pye Unicam SP1800 ultravioletrecording spectrophotometer operated at a wavelength of 540 nm and a slit height of 2 mm.  296 C. D.  Ockleford  and A. Whyte   • - .  \S-  /•W ''-•-^•J* ** SJM  Coated vesicles of human placenta  297 RESULTS Transmission electron microscopy of sectioned material Coated vesicles occur at or near the cell surface of the syncytiotrophoblast of thehuman placenta. They are frequently observed near microvillous areas of the cellsurface (Figs, i, 2) but are rare enough to have escaped detection in areas underlyingcell surface which is devoid of microvilli. The microvillous border of the cell ispopulated with profiles of uncoated vesicles of similar size to the profiles of coatedvesicles (Fig. 2). The microvilli and the surface region of the cell contain actin-likemicrofilaments with a diameter of about 9 nm. The rather disordered nature of thesemicrovilli compared for example with those of intestine is reflected at the macro-molecular level (Fig. 2). The microfilaments in placental microvilli are not packed inparallel in the regular hexagonal arrays seen in microvilli of intestine. Immediatelybelow the cell surface of the syncytiotrophoblast, which  in vivo  is in contact withmaternal blood, are a number of microtubules about 24 nm in diameter. Data on thedistribution of coated vesicles relative to the nearest cell surface are presented inTable  1  and in Figs. 3-5. These illustrate the fact that a very large proportion (atleast  89  ) of coated vesicles lie less than 540 nm from the cell surface. The distance540 nm was chosen because it is the longest axis across the largest coated vesicle yetmeasured (Ockleford, 1976). The fact that a higher proportion of vesicles are indirect contact with the cell surface than are present in the cytoplasm immediatelybelow the surface might suggest that the process of vesicle formation is slow comparedwith the rate of their subsequent movement away from the cell surface.The image of the polygonally patterned structure on the surface of coated vesiclesobtained using tannic acid-fixed material was less informative than that obtained usingconventional fixation techniques. The electron-dense product surrounding the vesicleswas much thicker than the height of the ridges of the polygonally patterned lattice.The 3 cell surface glycocalyx stains all reacted positively with the maternal surfaceof the syncytiotrophoblast (Figs. 1, 6, 7). However, the electron-dense regions wereconsiderably wider in the lumen of vesicles and in the caveolae of forming vesicles thanelsewhere on the surface when both ruthenium red (Fig. 6) and Alcian blue (Fig. 7)were used as stains. Within vesicles and caveolae this layer was up to about 40 nm inwidth and sometimes completely occluded the lumen of the vesicle. On other areasof the cell surface the layer of stain was usually less than 10 nm wide. Staining withcolloidal iron hydroxide at low pH was positive and remained so after neuraminidasedigestion, after esterification and after esterification followed by saponification.The potassium pyroantimonate cation-precipitation technique produced a rather Fig. 1. Transmission electron micrograph of a transverse section through a hollowfinger-shaped chorionic villus stained with Alcian blue. The outermost layer ofcytoplasm is the continuous syncytiotrophoblast (s). In places this contacts the base-ment membrane  bm).  An incomplete layer is formed of pale-staining cytotrophoblastcells (c) which are frequently interposed between the basement membrane and thesyncytiotrophoblast. The lumen (/) of the villus is for the most part structureless butcontains occasional mesenchymal cells  m)  and developing blood vessels  bv)  x 2400.
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