Ultrastructural pathologic changes of rat extraembryonic visceral endodermal cells exposed to teratogenic antibodies in vivo

It has been well established that certain heterologous tissue antibodies may induce abnormal embryonic development when injected into pregnant rodents during the organogenetic period. It has been postulated that these antibodies indirectly cause embryopathy by interfering with the normal functions of the yolk-sac placenta. The exact mechanism whereby these antibodies may induce placental pathology is not known. Specific teratogenic antibodies against a homogeneous rat kidney glycoprotein or a visceral yolk-sac glycoprotein antigen were injected intraperitoneally into 9th day pregnant rats. Electron microscopic examinations of the extraembryonic visceral endodermal cells of the egg cylinder were performed at 4, 6, 9, and 24 hours after the administration of the teratogenic antibodies. Control animals were injected with normal rabbit serum proteins. Extraembryonic visceral endodermal cells were similarly processed and examined as the experimental groups. The results seemed to indicate that the teratogenic antibodies induced increased autophagocytosis and morphologic changes associated with the phagolysosomes (secondary lysosomes) within the extraembryonic visceral endodermal cells at 9 hours following antibody administration. After 24 hours there was an apparent reduction or a complete disappearance of the supranuclear phagolysosome-like and lysosome-like structures, and the appearance of many large and small electron lucent vacuoles containing finely granular materials. Similar ultrastructural pathology was not observed in the 4 and 6 hour experimental and all of the control groups of animals. No other obvious intracellular or intercellular changes were observed in all of the experimental groups. Although the exact mechanism whereby the teratogenic antibodies may induce pathologic changes in the extraembryonic visceral endodermal cells remains to be determined, the present ultrastructural study demonstrated, for the first time, that teratogenic antibodies induced abnormal pathology in the extraembryonic visceral endodermal cells during the critical period of organogenesis.     

Abnormal embryonic development induced by antibodies to rat visceral yolk-sac endoderm: isolation of the antigen and localization to microvillar membrane

An antigenic substance was isolated from rat visceral yolk-sac endoderm of the 18th-20th days of gestation by extraction with the nonionic detergent Nonidet P-40, Sephacryl S-300 gel filtration, and Ricinus communis agglutinin affinity chromatography. The rabbit antiserum directed against this antigenic substance when injected into pregnant rats during the period of organogenesis caused abnormal embryonic development, fetal growth retardation, and embryonic death. Ouchterlony gel diffusion analysis demonstrated that the antiserum formed one immunoprecipitin band against the crude detergent extract and a complete identity between the present visceral yolk-sac antigen and the renal glycoprotein antigen previously isolated (C. C. K. Leung, (1982) J. Exp. Med. 156, 372-384). The antigen eluted from the antibody affinity column appeared to consist of two major peptides of 60 and 30 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Indirect immunofluorescent and immunoperoxidase localization studies at the light microscopic level demonstrated that both rat renal proximal tubule and embryonic visceral yolk-sac endoderm at various gestational stages (including the organogenetic period) shared the same antigen. Indirect immunoperoxidase localization studies at the electron microscopic level demonstrated that the antigen was a part of (or associated with) the microvillar membrane and membrane invaginations at the base of the microvilli of the renal proximal tubule and visceral yolk-sac endoderm. In vivo immunoperoxidase localization studies demonstrated that the teratogenic antibodies localized within the large phagolysosomes and the apical vesicles of the visceral yolk-sac endoderm. It is postulated that visceral yolk-sac pathology was induced by the antibodies.     

Passive Heymann nephritis induced by rabbit antiserum to membrane antigens isolated from rat visceral yolk-sac microvilli

Passive Heymann nephritis (PHN) is an animal model of immune-complex-induced renal disease resembling human membranous glomerulonephritis. It was induced in rats by injecting rabbit antiserum directed against glycoprotein antigens isolated from rat embryonic visceral yolk-sac microvilli (VYS-MV). The glycoprotein antigens were isolated by extracting the VYS-MV with detergent Nonidet P-40 followed by gel filtration in Sephacryl S-300 and finally by lectin affinity chromatography with Ricinus communis agglutinin I. In vitro immunofluorescent localization studies demonstrated that the nephritogenic antibodies were localized along the apical region of the visceral yolk-sac endodermal cells and the brush border of the proximal tubular cells of the kidney. Rats injected with a single dose of the antiserum manifested proteinuria. Indirect immunofluorescent studies showed that the injected rabbit IgG was localized in vivo along the capillary walls of the glomerulus in a granular fashion. Electron microscopic examination of the same kidney glomeruli revealed numerous electron-dense deposits along the lamina rara externa of the glomerular basement membrane. Fusion of the epithelial foot processes was also present. These findings represent the typical immunopathological characteristics of Heymann nephritis. Furthermore, with the aid of Ouchterlony analysis, the antiserum against the isolated VYS antigens exhibited an immunoprecipitin band which was in common with that formed by the antiserum against the homogeneous nephritogenic antigen (gp330) of renal brush border origin. Thus, the nephritogenic antigens which have been found to be associated with the brush border of the renal proximal tubules may also be present or cross-reacted in the microvilli of the rat embryonic visceral yolk-sac.     

Effects of teratogenic antibodies on cultured visceral yolk-sac endodermal cells: cytotoxicity and morphological studies

Primary cultures of visceral yolk-sac (VYS) endodermal cells were used to assess the effects of teratogenic and nonteratogenic antibodies. When assessed by cytotoxicity assay, teratogenic antibodies appeared to be lethal to the cultured cells at high concentrations (1.25-5 mg of antibodies per ml of culture medium). At a nonlethal dosage, the teratogenic antibodies induced morphological changes, including retraction and rounding up of living cells. The cytotoxic effect as well as the effect on cell morphology appeared to be dose-dependent and specific to VYS endodermal cells. The mechanisms of cell killing were not the same as those attributed to complement-mediated cell lysis. The nonteratogenic antibodies did not have any cytotoxic effect nor did they cause any cell morphological alterations. The results of this investigation, when interpreted by correlating the dose-dependent effects of the teratogenic antibodies on cultured endodermal cells with the in vivo teratogenic effect, suggest that teratogenic antibodies when given at a teratogenic dose cause congenital abnormalities without killing the VYS endodermal cells.     

Development, morphology, and function of the yolk-sac placenta of laboratory rodents

A review of current knowledge of the unusual structure and several functions of the yolk-sac membranes of common laboratory rodents, viz., rats, mice, hamsters, guinea pigs and gerbils, enables a better assessment of the significance of this maternofetal exchange system in the experimental production of congenital anomalies. The anatomy of both visceral and parietal walls of the rodent yolk-sac placenta--specifically the anatomical relationships of each wall with maternal and with other fetal tissues--depends on the mode of origin and subsequent development of the yolk sac in these several species. Accordingly, the developmental biology of the rodent yolk sac is described. Since both fine structure and anatomical relationships also determine in large measure the functioning of the membrane as a whole in the absorption of selected materials either for intracellular digestion or for cellular translocation and transport to the developing embryo, the anatomy of the yolk sac is considered in detail. Similarly, since available evidence strongly suggests that teratogenic agents induce perturbations in the cellular mechanisms that control these several functions of the yolk-sac placental system in the production of birth defects, additionally an account is given of the cell biology of the membrane, i.e., endocytosis and targeting/trafficking of materials either for digestion within the epithelium at the maternal surface of the visceral yolk sac or for translocation across the yolk-sac membrane as a whole.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24 h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4 degrees C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5-60 min at 20 degrees and 37 degrees C in Con-A-free serum resulted in a temperature-dependent internalization of membrane-bound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37 degrees C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constituents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

Summary The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4°C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5 60 min at 20° and 37°C in Con-A-free serum resulted in a temperature-dependent internalization of membranebound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37°C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constitutents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Dynamics of antibody transfer from mother to fetus through the yolk-sac cells in the rat

In rodents, maternal immunoglobulins are transported intact by the yolk-sac visceral epithelium from mother to fetus. The main purpose of the present paper is to study the dynamics of the uptake and transport of immunoglobulins by the rat yolk-sac using a new experimental design. The results show the rapid binding of IgG to the cell membrane microvilli since only 30 sec were sufficient for this attachment to occur. The endocytic process also appears to be very fast as localization of IgG in clusters, pits and microvesicles were observed after 5 min of contact between the yolk-sac and the IgG solution. Moreover, the antibodies were detected in the intracellular spaces within 15 min of incubation.     

Fine structural analysis of the effect of trypan blue on the visceral endoderm of the mouse egg cylinder

The effects of a maternal injection of trypan blue on primitive streak mouse embryos were studied with electron microscopy. Commercial trypan blue was purified by descending paper chromatography, and pregnant females received an intraperitoneal injection of the collected blue fraction on the evening of the 7th day of gestation. Ultrastructurally, the changes in the visceral endoderm were apparent 10 min after the injection and included an increase in the number of fuzzy-coated vesicles in the apical cytoplasm. By 20 min the apical cytoplasm of the extraembryonic visceral endodermal cells was filled with many fuzzy-coated vesicles and numerous vacuoles of various size and electron density. 30 min after the injection, the extraembryonic visceral endodermal cells were relatively smooth lacking a microvillous border and evidence of endocytic activity was rare. Many embryonic visceral endodermal cells were observed in various stages of degeneration although the underlying embryonic ectoderm appeared unaffected. Morphologically, it appears that trypan blue exerts its first effect by altering the endocytic activity of the visceral endoderm.     

The annular hematoma of the shrew yolk-sac placenta

The annular hematoma of the shrew, Blarina brevicauda, is a specialized portion of the yolk-sac wall. In this study, we have examined the fine structure of the different cellular components of the anular hematoma. Small pieces of the gestation sacs from seven pregnant shrews were fixed in glutaraldehyde and osmium tetroxide and processed for transmission electron microscopy. In the area of the trophoblastic curtain, the maternal capillary endothelial cells were hypertrophied and syncytial trophoblast surrounded the capillaries. Cellular trophoblast covered part of the luminal surface of the curtain region, whereas masses of apparently degenerating syncytium were present on other areas of the surface. Maternal erythrocytes, released into the uterine lumen from the curtain region, were phagocytized and degraded by the columnar cells of the trophoblastic annulus. No evidence of iron or pigment accumulation was evident in the parietal endodermal cells underlying the annular trophoblast. Parietal endodermal cells were characterized by cuboidal shape, widely dilated intercellular spaces, and cytoplasm containing granular endoplasmic reticulum. Endodermal cells of the visceral yolk-sac accumulated large numbers of electron-dense granules as well as glycogen in their cytoplasm. Hemopoietic areas and vitelline capillaries were found subjacent to the visceral endoderm. The various portions of the yolk-sac wall of Blarina appear to perform complementary functions which are probably important in maternal-fetal iron transfer.     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

Preparation of IgG-binding membrane vesicles from the microvillar brush border of fetal rabbit yolk sac.

A one step procedure is described for the production of membrane vesicles from the endodermal microvillar brush border of the fetal rabbit yolk sac splanchnopleur. The vesicles, examined by light and electron microscopy, were shown to consist of biomolecular leaflet unit membrane, coated to varying extents with glycocalyx. By fluorescence microscopy, the homologous immunoglobulin, FITC-IgGR, has been shown to bind to the glycocalyx-coated vesicles as well as the glycocalyx-coated brush border of the intact yolk sac, whereas, the heterologous bovine immunoglobulin, FITC-IgGB, fails to bind under comparable conditions. These observations demonstrate the specificity of the receptors for the homologous IgG.     

Maternal transferrin uptake by and transfer across the visceral yolk sac of the early postimplantation rat conceptus in vitro

Uptake and transfer of maternal transferrin by rat embryos during organogenesis in vitro was investigated using radiolabelled rat transferrin and rocket immunoelectrophoresis. Colloidal gold to which rat transferrin was adsorbed was used as an electron microscopical marker in order to follow the route taken by internalised transferrin across the visceral yolk sac. Culture of rat conceptuses from 9.5 to 11.5 days of gestation in rat or human sera resulted in the passage of rat or human transferrin from the culture medium into the extraembryonic coelom as determined by quantitative immunoelectrophoretic analysis of exo-coelomic fluid. The concentration of human transferrin which was transferred to the exo-coelomic fluid of conceptuses cultured in whole human serum at 10.5 days and 11.5 days of gestation was similar to the concentration of rat transferrin in the fluid of conceptuses cultured in rat serum which had been diluted with Hanks' saline to 50% in order to match the levels of transferrin found in human serum. Growth of rat embryos in 50% rat serum was identical to embryonic growth in 100% rat serum. Uptake of radiolabelled rat transferrin by the visceral yolk sac at 11.5 days of gestation, following culture for 60 min in radiolabelled medium, was much greater than nonspecific uptake of radiolabelled bovine serum albumin. Accumulation of radiolabelled transferrin by the embryo was reduced by the inclusion of unlabelled transferrin into the culture medium. Uptake of transferrin adsorbed 18 nm gold particles was mediated by attachment to coated pits on the apical cell surface of the extraembryonic endoderm. Transferrin-adsorbed gold colloid was internalised via coated vesicles and found in cisternal structures of the peripheral and juxtanuclear areas, as well as in smooth and coated vesicles deep within the cell. The intercellular presence of gold particles in the endodermal layer of the visceral yolk sac and their presence in the mesoderm after 60 min of incubation suggested that passage of transferrin was rapid and mediated by vesicular evagination from the extraembryonic endoderm. These findings suggest that maternal transferrin is the primary source of transferrin for the early rat embryo and its passage to the exo-coelom and embryo is mediated by specific receptors on the apical surface of the extraembryonic endoderm.     

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Demonstration of oestrogens in developing pig trophectoderm and yolk sac endoderm between days 10 and 16

Segments of individual blastocysts collected on Days 10, 12, 14 and 16 were examined microscopically to observe yolk-sac development and treated immunocytochemically to localize oestrogens in specific membranes. Mesoderm was present beneath the embryonic disc of ovoid blastocysts on Day 12. The mesoderm spread beyond 1 cm from the disc on Day 14, producing a splanchnic yolk-sac membrane extending across the blastocoelomic cavity, but no mesodermal cells had yet reached 5 cm. By Day 16, proliferation of mesoderm and development of the yolk sac had progressed beyond 20 cm from the disc in most of the specimens examined. Incubation of ultrathin sections with sheep antiserum to oestrone or oestradiol-17 beta followed by rabbit anti-ovine IgG-gold complex and subsequent counting of gold particles retained over the tissues gave a weakly positive reaction for oestrone in trophectodermal cells on Day 10. The most intense reaction for oestradiol-17 beta was also present in the trophectoderm and yolk-sac endoderm on Days 12, 14 and 16.     

Effects of L-asparaginase on rat embryonic development and yolk sac membrane in vitro

A comparative analysis of the teratogenic effects of L-asparaginase on 10.5- and 11.5-day rat embryos after 24 and 48 hours of exposure in vitro, respectively, were performed. Several medium concentrations of L-asparaginase (0.05, 0.25, and 1.5 IU/ml) were tested in both embryo series. Resulting embryos were submitted to morphological studies in a search for a specific route of pathogenesis. Morphological alterations of the visceral yolk sac were also studied to investigate its contribution to L-asparaginase teratogenicity in rats. Main embryonic malformations (open truncal neural tube, open encephalic vesicles, anophthalmia, lack of inversion, abnormal frontolateral protrusions, great vascular dilations at the cephalic level) and developmental retardation were already generated after the first 24 hours of culture (embryos of 10.5 days) and presented a dose-response relationship. Vascular dilations and neurulation disturbances seemed to be related to an early mesenchyme deficiency. Reduced number of mesenchymal cells was more evident in embryos of 10.5 days than those of 11.5 days, suggesting the existence of a later compensatory mechanism of cellular proliferation in the older embryo. Visceral yolk-sac endodermal cells at both embryonic stages were greatly deformed and enlarged by an increase of the high electron-dense vacuolar system. Therefore, both a blockage of the processes of lysosomal digestion and derived trophic deficiencies probably existed. A double teratogenic mechanism for L-asparaginase is postulated: a direct action mainly in younger embryos (before invagination of the embryo into the yolk sac) and a yolk sac-mediated one.     

Simultaneous demonstration of two antigens in ultrathin cryosections by a novel application of an immunogold staining method using primary antibodies from the same species

A recently developed immunocytochemical double-staining method for ultrathin Epon and Lowicryl K4M sections has been adopted for use on ultrathin cryosections. The essential features of the method include: staining for the first antigen by the indirect method using sufficient concentrations of second antibodies conjugated to colloidal gold particles to saturate available epitopes on the primary antibodies; supporting the cryosections by methyl cellulose followed by paraformaldehyde vapour treatment (30-60 min at 80 degrees C); removal of the methyl cellulose followed by staining for the second antigen using primary antiserum from the same species and another size class of colloidal gold particles conjugated to second antibodies. Contaminating staining does not occur if the paraformaldehyde vapour treatment exceeds 30 min, as this treatment destroys the combining sites on the second antibodies applied in the first staining cycle. Successful double-staining was documented using primary rabbit antibodies to growth hormone and corticotropin and anti-rabbit IgG conjugated to 5 and 15 nm colloidal gold particles. Following double-staining, the ultrathin cryosections may be silver-enhanced to improve detectability of the markers at low magnification.     

Cell surface labelling of mononuclear cells with antisera associated to turnip yellow mosaic virus or alphalpha mosaic virus particles. A freeze-etch study

Synopsis Turnip yellow mosaic virus (TYMV) and alphalpha mosaic virus (AMV) were used as immuno-electron microscopical markers to detect cell surface receptors on mononuclear cells in freeze-etch replicas. TYMV particles were conjugated with vacuum-distilled glutaraldehyde to rabbit IgG anti-mouse immunoglobulins (TYMV-RAMIg conjugate) or to rabbit IgG anti-mouse antigen (TYMV-RAMTh conjugate). B-lymphocytes incubated with TYMV-RAMIg conjugate showed either randomly distributed particles or patches of virus particles on the etched surface of the cell membrane. Mouse thymocytes incubated with TYMV-RAMTh conjugate, however, showed only a random distribution of the virus particles. Human mononuclear cells incubated with rabbit IgG anti-AMV and AMV for the demonstration of the receptors for the Fc fragment of IgG showed the oblong shape of the AMV particles on the etched cell membrane. Fc receptors were either randomly distributed or aggregrated into patches. It is concluded that both types of virus particles are useful markers for the demonstration of membrane receptors in freeze-etch replicas of labelled cells.     

Simultaneous demonstration of two antigens in ultrathin cryosections by a novel application of an immunogold staining method using primary antibodies from the same species

Summary A recently developed immunocytochemical double-staining method for ultrathin Epon and Lowicryl K4M sections has been adopted for use on ultrathin cryosections. The essential features of the method include: staining for the first antigen by the indirect method using sufficient concentrations of second antibodies conjugated to colloidal gold particles to saturate available epitopes on the primary antibodies; supporting the cryosections by methyl cellulose followed by paraformaldehyde vapour treatment (30–60 min at 80°C); removal of the methyl cellulose followed by staining for the second antigen using primary antiserum from the same species and another size class of colloidal gold particles conjugated to second antibodies. Contaminating staining does not occur if the paraformaldehyde vapour treatment exceeds 30 min, as this treatment destroys the combining sites on the second antibodies applied in the first staining cycle. Succesful double-staining was documented using primary rabbit antibodies to growth hormone and corticotropin and anti-rabbit IgG conjugated to 5 and 15 nm colloidal gold particles. Following double-staining, the ultrathin cryosections may be silver-enhanced to improve detectability of the markers at low magnification.     

Changes in plasma-membrane-associated filaments during endocytosis and exocytosis in polymorphonuclear leukocytes

We examined the filaments associated with the cytoplasmic surface of the plasma membrane in rabbit exudate PMNs during phagocytosis of particles, or during “frustrated phagocytosis” with exocytosis of storage granules. Cells were plated onto yeast particles glued to coverslips with polylysine or onto coverslips coated with sheets of heat-agglutinated IgG. After periods ranging from 1 to 15 min, we disrupted the cells by a jet of salt solution and exposed their inner membranes. These broken cells were fixed immediately and processed for SEM. Whole cells were also prepared for SEM or TEM. At the site of PMN adherence to an opsonized yeast particle, a network of globular centers and thin, branched filaments appears on the cytoplasmic surface of the plasma membrane, while the outstretching lamellipodia contain a mesh of such filaments but no globular centers. Within 1 to 2 minutes, these structures disappear from the invaginating portion of the developing vacuole, and the cell's storage granules fuse with the barren membrane regions. These activities occur in rapid sequence over the vacuolar membrane after the first contact, until the phagocytosed particle is wholly encircled by a smooth, loose membrane, separated from the cell surface. A comparable filament pattern or complex was seen during “frustrated phagocytosis” on IgG sheets. At times between 1 and 5 min after plating, the cytoplasmic surfaces of these adherent membranes contain denuded central regions and peripheral nets of globular centers with radiating, thin, branched filaments. Granules apparently fuse with the bare areas. Thus we have obtained evidence of filament association with the plasma membrane at sites of adherence (to phagocytosable or nonphagocytosable surfaces) and have traced the subsequent disappearance of the filaments with degranulation.