Amorphous Extracellular Material Research Articles

Hyalinizing spindle cell tumor with giant rosettes: a report of three cases with ultrastructural analysis.

We report the light microscopic, ultrastructural appearance and immunohistochemical staining profile of three distinctive soft-tissue tumors recently designated hyalinizing spindle cell tumor with giant rosettes. The tumors occurred in two men, 41 and 54 years old, and one woman, 62 years old. Two tumors arose in the lower extremities and one in the upper arm. Two tumors were resected and measured 3 and 13.2 cm in greatest diameter; a biopsy only was done of the third tumor. Grossly, the tumors had a tan, pink, or white cut surface. The largest tumor exhibited central cystic change. Microscopically, they all displayed similar features and were composed of fibromyxoid regions, with areas of hyalinization in two tumors and focal ossification in one tumor. Scattered throughout each of the tumors were rosette-like structures in which neoplastic cells were arranged around a central collagenous core. Ultrastructurally, the neoplastic cells demonstrated the features of fibroblasts. In all tumors, there was abundant extracellular collagen fibers and in one there were large aggregates of amorphous extracellular external lamina-like material. The center of the rosette-like structures was composed of banded collagen fibers and the cells at the periphery of the rosettes had ultrastructural features similar to the neoplastic spindle cells located elsewhere in the tumor. Immunohistochemically, the tumor cells stained for vimentin. There was focal staining of the widely distributed spindle cells and cells that formed the rosettes for Leu-7, S-100 protein, and CD34. In one tumor, there was faint diffuse staining of the spindle cells for neuron-specific enolase. One tumor (with the amorphous extracellular material) stained for type IV collagen. There was no staining for desmin, muscle actin, smooth muscle actin, keratin, or epithelial membrane antigen. These results demonstrate that hyalinizing spindle cell tumor with giant rosettes is composed of fibroblasts. We did not demonstrate any ultrastructural or immunohistochemical differences between the spindle cells that comprised the majority of the mass and those that surrounded the rosette-like structures. There was no ultrastructural evidence of neural differentiation to explain the focal S-100 protein and Leu-7 staining of the tumor cells.

Wells' syndrome triggered by centipede bite.

A 68-year-old African–American woman was awoken from her sleep by a centipede bite on the dorsum of her left hand. Several days later, the patient presented to the emergency room complaining of fevers, severe itching, swelling, and blistering (Fig. 1) of both hands. The past history was unremarkable. There was no history of asthma or use of medications. Physical examination at that time was consistent with a bullous cellulitis of the hands. Laboratory investigations revealed: white blood cell count (WBC), 10.3 (normal, 41, 2, 3-9, 10.5); differential: 54 neutrophils, 15 lymphocytes, and 31 eosinophils. The platelet count was 225,000/mm3. A blood chemistry profile was within normal limits. Blood cultures and stool examination for ova and parasites were negative. The patient was treated with oral cephalexin and diphenhydramine. Figure 1Open in figure viewerPowerPoint Left hand reveals a bulla and several vesicles at the site of the centipede bite Two weeks later the patient was seen in the dermatology clinic. She complained of unabated pruritus and extensive new lesions. Physical examination revealed a moderately obese woman with normal vital signs. Firm gray plaques with an irregular contour were present on the left hand. The buttocks (Fig. 2) and extensor aspects of her upper arms were strikingly indurated and had a blue–gray discoloration. Violaceous, indurated oval patches were noted on the medial aspect of both knees. Finally, a slate-colored, irregularly outlined plaque on the heel of the right foot was present. Complete blood count revealed: WBC, 16.5; differential, 8 neutrophils, 9 lymphocytes, 81 eosinophils, and 2 monocytes. The differential was repeated and confirmed. The platelet count was 568,000/mm3. Figure 2Open in figure viewerPowerPoint Blue–gray discoloration and induration of the buttocks as well as the proximal thighs is seen. These changes became apparent several weeks after the initial blistering reaction of the hands A skin biopsy from the patient's left upper arm revealed a superficial and deep perivascular dermatitis. In addition, the dermis had a dense inflammatory infiltrate consisting of numerous interstitial eosinophils, many of which were degranulated. Collagen bundles coated with eosinophilic granules characteristic of flame figures were abundant (Fig. 3). The changes were consistent with eosinophilic cellulitis. Chest X-ray revealed no evidence of infiltrates. Figure 3Open in figure viewerPowerPoint There is a dense infiltration of eosinophils in the dermis, many of which have degranulated. Several collagen bundles coated by an extracellular amorphous material (flame figures, arrow) are evident in the center (hematoxylin and eosin stain; original magnification, ×100) A course of oral corticosteroids was prescribed. One week later, the lesions were substantially less indurated, but otherwise similar in appearance. Complete blood count revealed: WBC, 9.9; differential: 21 neutrophils, 19 lymphocytes, 1 basophil, 51 eosinophils, and 8 monocytes. The platelet count was 408,000/mm3. Hematologic consultation was arranged for bone marrow biopsy and examination. Unfortunately, the patient did not return for subsequent appointments.

Abnormal extracellular material in the levator palpebrae superioris complex in congenital ptosis.

To determine the pathologic abnormalities of the levator palpebrae superioris in congenital ptosis. By means of light and electron microscopy and immunohistochemistry, anterior levator tissue specimen from 15 patients with congenital ptosis excised during routine levator resections were examined. All specimens showed lack of muscle fibers with endomysial and perimysial fibrosis and thickening of the aponeurosis. In addition, in four of the 15 patients, an abnormal extracellular material was present. By light microscopy this appeared as an unusual amorphous material arranged in clumps and bands, and electron microscopy showed it to consist of parallel coarse bundles of fibrillogranular material. Collagen type III and fibronectin were identified within this material by immunohistochemistry. There was no detectable collagen types I, II, IV, V, VI, or VII or laminin, and the material did not stain for actin, myosin, myoglobin, amyloid P component, or amyloid A. In four of 15 samples of levator palpebrae superioris from patients with congenital ptosis, we identified an unusual amorphous extracellular material that stained positively for collagen type III and fibronectin on immunohistochemistry. This novel material, which we call "amorphocollagenoid," may represent a product of dysgenesis of the levator tissues. The source and full composition of this material merit further study.

Characteristics of Actinobacillus actinomycetemcomitans invasion of and adhesion to cultured epithelial cells.

Actinobacillus actinomycetemcomitans (A.a.) is highly implicated in periodontitis. We have developed several in vitro models using the KB oral cell line to examine A.a.-epithelial cell interactions. In support of the use of KB cell line model systems is our finding that A.a. invaded KB and primary gingival cells to the same extent. Invasion is an active event which requires new protein synthesis by both KB and A.a. Like many other intracellular parasites, A.a. invade by receptor-mediated endocytosis. We observed that internalized A.a. were surrounded by foci of actin which had been transported from the periphery of the KB cell. Adhesion of A.a. to KB cells occurred rapidly and stimulated the formation of microvilli. Adhesion is affected by both host factors (saliva, serum, [NaCl]) and culture conditions. Multiple determinants [fimbriae, outer membrane proteins, vesicles, and/or an extracellular amorphous material (ExAmMat)] which are either associated with the A.a. surface or are released into the milieu are involved. We determined that ExAmMat can convey adhesiveness to weakly adherent A.a. and to at least one other oral species (Streptococcus parasanguis).

The surface of Erysiphe graminis and the production of extracellular material at the fungus – host interface during germling and colony development

Low temperature scanning electron microscopy was used to study the development of Erysiphe graminis DC f.sp. avenae Marchal from germination through infection to sporulation of the fungal colony. To clarify resolution of the fungus–host interface and facilitate interpretation of fungal surface structures, epicuticular waxes were removed from host leaves before inoculation. Whereas conidia were covered in spinelike protrusions or globular bodies, young germ tubes, appressoria, and hyphae were initially smooth walled, but by 15 h after inoculation, wartlike bodies, resembling globular bodies on conidia, were present on first appressorial lobes; these increased in number, eventually covering the appressorium surface and appearing on contiguous hyphae. Wartlike bodies also appeared at junctions of hyphal branches, on hyphal appressoria, and on conidiophore basal cells. Their function, if any, is unknown. The meristematic zone, at the apex of the conidiophore basal cell, remained smooth walled, but globular bodies appeared on the wall of young conidia as soon as the limiting septum had formed. Observations with the fungus in situ revealed the presence of amorphous extracellular material around primary germ tubes and appressorial lobes. Extracellular material was also present beneath appressorial germ tubes and hyphae but it was hidden unless the fungus was displaced. It could not be seen beneath conidia. The extracellular material appeared to be adhesive, sticking the fungus firmly to the host surface. Removal of the fungus showed that the extracellular material was deposited close to the tip of developing germ tubes and hyphae. It was particularly thick around primary germ tubes and appressorial lobes, and a discrete ring of extracellular material was often visible around penetration pores (holes in the leaf surface seen beneath primary germ tubes and appressorial lobes). In addition to its adhesive properties, the extracellular material may act as a matrix in which fungal enzymes are sited and focused for attack on the host. Key words: Erysiphe graminis, low temperature scanning electron microscopy, extracellular material, fungal adhesion, fungal surface morphology.

Characteristics of adherence of Actinobacillus actinomycetemcomitans to epithelial cells

Actinobacillus actinomycetemcomitans smooth variants [SUNY 75(S), SUNY 465, 652] were investigated for their ability to adhere to KB epithelial cells. Both the type of medium (broth versus agar) and anaerobicity influenced adherence levels and cell surface characteristics. Optimal adherence was observed with all three strains after growth of the bacterial cells in broth under anaerobic conditions, a condition which was associated with extracellular microvesicles. Adherence of SUNY 75(S) also was correlated with extracellular amorphous material, whereas adherence of SUNY 465 was also associated with fimbriation which accompanied a smooth to rough phenotype shift. The relationship between adherence and extracellular vesicles, extracellular amorphous material, and fimbriation suggests that all of these components may function in A. actinomycetemcomitans adherence to epithelial cells. The phenotype shift observed in SUNY 465 cells is further evidence that A. actinomycetemcomitans SUNY 465 is predisposed to variant shifts which are associated with changes in adherence and invasion properties.

Assessment of the long-term corneal response to hydrogel intrastromal lenses implanted in monkey eyes for up to five years

The biocompatibility of hydrogel intracorneal lenses (ICLs) implanted in monkey eyes was evaluated for periods ranging up to five years. Seventy-three plus or minus powered ICLs made of Lidofilcon A (68% water) or Lidofilcon B (79% water) were implanted following lamellar dissection with a microkeratome. Ten sham surgical procedures were performed without ICL implantation as controls. Eyes were followed for up to five years by slitlamp biomicroscopy and specular microscopy. Light and transmission electron microscopic evaluations of enucleated eyes were performed at various intervals. Minimal tissue reaction was noted; both hydrogel materials appeared to be equally well tolerated. Failures usually occurred as a result of microkeratome problems encountered during surgery. Histopathological changes to the cornea included epithelial thinning anterior to the thickest portion of the ICL, fibroblastic activity along the ICL-stromal interface, and deposition of an amorphous extracellular material adjacent to the ICL. These observations did not appear to be clinically significant as the eyes were quiet by slitlamp examination. Removal of three ICLs eight to ten months prior to enucleation restored the normal histological characteristics of the cornea. The endothelial cell density of ICL-implanted eyes decreased by 4.3% (n =17) six months after surgery but remained stable thereafter. The variation in endothelial cell area and percentage of hexagonal cells did not change over 50 months. The results appear to demonstrate that high water content synthetic ICLs can be well tolerated in the monkey cornea for up to five years.

Interactions between extracellular Borrelia burgdorferi proteins and non-Borrelia-directed immunoglobulin M antibodies

Previous work showed that outer surface protein A (OspA) and OspB of Borrelia burgdorferi may occur within an extracellular multiprotein complex, which was resolved by electrophoresis as an 83-kDa major extracellular protein band. To characterize the 83-kDa band, we sequenced the N terminus of the predominant peptide in the band and examined the interaction between the associated proteins. Peptide sequence and amino acid composition comparisons showed identity with the heavy chain of immunoglobulin M (IgM). Reduction sensitivity experiments and the recognition of the band by antibodies specific for rabbit mu chain indicated that the multiprotein complex contained pentameric IgM. Immunoelectron microscopy showed that anti-mu chain antibodies and monoclonal antibodies to OspA and OspB bound to extracellular amorphous material surrounding cells. Furthermore, the Osps coprecipitated with either nonspecific polyclonal rabbit IgM antibodies or with murine monoclonal anti-human serum albumin IgM antibodies, using insoluble anti-mu chain antibody conjugates. Although the apparent 83-kDa complex was stable under conditions of chelation and concentrated salts, it was disrupted by treatment with neuraminidase. These results indicate that extracellular B. burgdorferi proteins, including OspA and OspB, interact with IgM. The association is apparently not a classic antibody-antigen interaction but may result from other mechanisms.

Development of the notochord in normal and malformed human embryos and fetuses.

In view of its possible involvement in early embryogenesis and teratogenesis, the developmental characteristics of the human notochord were studied by light and electron microscopy and immunohistochemistry on 20 human conceptuses (5th-22nd week). At the earliest embryonic stages examined, the notochord is closely related to both the pharyngeal endoderm and the neuroectoderm of the posterior (tail) end of the neural tube. In both regions the interspace is bridged by slender cytoplasmic processes, lined with basal lamina and filled with amorphous extracellular material containing collagen types III and IV and laminin. The notochordal cells express cytokeratin brightly and vimentin weakly. As embryonic age progresses, the notochord gradually separates from the epithelia, becomes the axis of developing spinal column and undergoes progressive cell degeneration and rearrangement within the vertebral bodies. This is associated with extensive production of extracellular material and the first appearance of fibronectin. Intracellularly, the expression of vimentin gradually increases, while that of cytokeratin slightly weakens. Changes in the notochord parallel other developmental events in axial organs. In anencephalic fetuses the course of the notochord is irregular and partly interrupted with segments outside the basichondrocranium in specimens with craniorachischisis.

Histochemical, ultrastructural, and electron microprobe analytical studies on the localization of calcium in rat incisor ameloblasts at early stage amelogenesis

The enamel organs of rat incisors were separated from the enamel surface and processed for rapid freezing and freeze-substitution. A histochemical stain for calcium (GBHA) of thick Epon sections revealed intense calcium reactions in the secretory ameloblasts, exclusively in the tubulovesicular structures extending throughout their distal cytoplasm. Electron microscopy revealed a thin layer of amorphous material with clusters of electron-dense granules along the distal surface of secretory ameloblasts. In young secretory ameloblasts without typical Tomes' processes, a considerable number of mitochondria were located in the distal cytoplasm and contained numerous electron-dense granules. Similar dense granules as well as fine ribbon-like electron-dense figures, all containing significant amounts of calcium, were observed in some of the tubulovesicular structures at the distal end of these cells. A putative exocytotic figure of such dense granules was also observed. The electron-dense granules were rare in more differentiated ameloblasts with elongated Tomes' processes, which occasionally displayed ribbon-like figures in some of the tubulovesicular structures in the process region. No significant calcium peak was detected in the extracellular amorphous material, secretory granules, or along the lateral plasma membranes. These observations may imply high calcium concentrations in mitochondria and tubulovesicular structures in the distal cytoplasm of secretory ameloblasts relative to that of the cytosol and support the possible contribution of these organelles in secretory ameloblasts to cellular calcium regulation at least in the early stage of amelogenesis.

Immunocytochemical in vivo localization of fibronectin‐rich contact sites on fibroblasts of normal periodontal ligament and inflamed gingiva

Fibroblast‐to‐matrix attachment sites were studied by routine electron microscopy and immunocytochemistry. Rabbit antibodies to beagle dog plasma fibronectin and sheep antirabbit antibodies conjugated with horseradish peroxidase or ferritin were used to localize fibronectin at fibroblast‐to‐matrix attachment sites. In fibroblasts of healthy periodontal ligament, the attachment sites consisted of rectangular patches of amorphous material juxtaposed to the external surface of the plasma membrane. At these sites, the cell membrane was more densely stained and the adjacent cytoplasm was characterized by increased density and a high concentration of cytoplasmic filaments. The extracellular plaques contained fibronectin. Morphometric analysis indicated that the attachment plaques were approximately 90 nm thick, 250 nm wide, and 550 nm long, and distributed uniformly over both the cell body and peripheral cytoplasmic processes. In inflamed gingiva, the attachment sites were larger, irregular in shape, and with greater amounts of extracellular amorphous material and fibronectin associated to the cell surface. Cytoplasmic filaments were more often bundled as stress fibers which terminated in fibronexus‐type junctions with extracellular fibronectin‐coated filaments.

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro.

Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyper- or hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.

Interaction of a Pyelonephritogenic Escherichia Coli Strain with the Tissue Components of the Mouse Urinary Tract

Immediately following the introduction of cells of a pyelonephritogenic strain of Escherichia coli (expressing both GS and MS adhesins) into the mouse bladder, these cells were found to be randomly distributed on the mucosal surfaces of both the bladder and the kidney. After 24hr. these adherent bacterial cells had proliferated to form nonrandomly distributed adherent microcolonies on both mucosal surfaces and large masses within the renal ducts. Large amounts of amorphous material, which we presume to be of both host and bacterial origin, was associated with the bacterial microcolonies on the infected tissues. The removal of urinary slime and tissue coat material by acid pretreatment increased both the degree and the apparent avidity of bacterial adhesion to the bladder mucosa. The adherent bacteria on the bladder mucosa were radically elongated, while those on the kidney mucosa usually retained their short rod-like morphological characteristics. These data suggest that pyelonephritogenic bacteria adhere to mucosal surfaces in both the bladder and kidney, and proliferate to form adherent microcolonies within which bacteria are associated with large amounts of amorphous extracellular material.

Ultrastructure of the dental epithelium during enameloid mineralization in a teleost fish, Cichlasoma cyanoguttatum

Secretory-stage inner dental epithilial cells (IDE) of tooth buds deposited an unmineralized, ectodermally-derived, enameloid collagen matrix. Pharyngeal plates bearing tooth buds were fixed: some were demineralized, others treated with guanidine-EDTA, then fixed and post-fixed in osmium tetroxide with potassium ferricyanide. Thin Epon sections were viewed in a Jeol 100B TEM. Nascent enameloid crystals were orientated parallel to the collagen fibres and attained widths of 200 nm. Enameloid collagen was absent in demineralized mature enameloid. The outer dental epithelial plasma membrane was deeply invaginated forming extensive channels associated with elongated fuzzy-coated vesicles. Four configurations of IDE cells were characterized by cellular constituents, including elongated granules, Golgi complexes, multivesicular bodies, large electron-dense granules and extracellular amorphous material which was also adjacent to cells containing few organelles associated with protein synthesis, within infoldings of ruffled apical membranes and multivesicular bodies. This material was considered to be resorbed enameloid collagen, not a secretory product.

Cumulus oophorus mucification during resumption of meiosis in the pig. A scanning electron microscope study.

The process of cumulus mucification in pig preovulatory follicles was examined by scanning electron microscopy. The localization of extracellular material and changes in the granulosa cell surface were observed at 0, 16, 20 and 40 h after hCG injection. At 0 h the pig oocyte cumulus complex was closely attached to the parietal layer of granulosa cells. Cumulus and parietal granulosa cells had microvilli and cytoplasmic projections connecting neighbouring cells. The network of extracellular amorphous material was observed for the first time at 16 h after hCG around the cells that formed a stalk between the parietal granulosa and the cumulus oophorus. At 20 h after hCG, the intercellular matrix was thicker and extended to almost all the cumulus oophorus surface; when visible, cells were often covered by blebs and ruffling membranes. All oocytes examined by light microscopy at 16 and 20 h after hCG were at the germinal vesicle stage with condensing bivalents. Therefore, it was concluded that oocyte nuclear maturation started at the same time that a mucified peduncle was forming between the cumulus oophorus and the parietal granulosa. Shortly before ovulation (40 h after hCG) the matrix material filled all intercellular spaces and mucification had extended to the corona cell layer and the zona pellucida surface.

Attachment of Staphylococcus aureus to polymethylmethacrylate increases its resistance to phagocytosis in foreign body infection.

The mechanisms responsible for the development of a pyogenic infection (most commonly due to staphylococci) in the vicinity of an implanted foreign body have been studied recently by several investigators. Thus, we have been able to demonstrate that the phagocytic function of residential polymorphonuclear leukocytes (PMN) is deficient in the presence of a foreign body. Others have shown that in the presence of foreign surfaces, microorganisms produce extracellular amorphous material, the pathogenic role of which is still to be defined. In the present study we use a novel assay system to demonstrate that Staphylococcus aureus Wood 46, after attachment to polymethylmethacrylate (PMMA), shows increased resistance to the phagocytic-bactericidal action of normal PMN. The first step of this assay involves the reproducible attachment of [3H]thymidine-labeled bacteria to PMMA cover slips. During the second step, attached bacteria were exposed to guinea pig peritoneal exudate PMN. In the third and final step, attached S. aureus cells were removed from the cover slips using a procedure harmless to the bacteria. The extent of bacterial detachment was estimated by radioactive counts and their viability by standard colony counts. Whereas bacteria that were attached artificially and rapidly by centrifugation and immediately exposed to PMN were killed in the phagocytic assay, bacteria adhering spontaneously to the cover slips for a prolonged period of time were more resistant to the killing action of the phagocytes. The spontaneous adherence of S. aureus to PMMA renders it poorly susceptible to the killing action of PMN.

Estudio ultraestructural de la mionecrosis inducida en raton por el veneno de terciopelo ( Bothrops asper) de Costa Rica

Skeletal muscle from white mice was examined one and three hr after i.m. injection of 50 μg of Bothrops asper venom. At the light microscopic level we observed nectoric fibers with myolitic appearance, as early as the first hr and more marked by the third hr. At the ultrastructural level three kinds of fibers were observed: (a) fibers with normal morphology, (b) fibers with slight alteration at the sarcoplasmic reticulum and (c) fibers with strong alterations in several organelles; in these fibers, the main pathological feature was the disruption of myofibril architecture. This disruption started with the disappearance of A, H, I and M bands, followed by the alteration of Z line. At the end of this process an amorphous mass of proteins was observed. Shortly following envenomation, sarcoplasmic reticulum showed moderate dilatation and proliferation, but when degenerative changes were more evident, smaller and more abundant vacuoles were seen. Also, the plasma membrane was altered and disrupted in some fibers, whereas many mitochondria showed a swollen appearance and, in some cells, they were disrupted. An amorphous extracellular material was also observed as a consequence of envenomation. It is proposed that the ultrastructural basis of the muscular necrosis induced by terciopelo venom is the disruption and severe alteration of the myofibril architecture.

Surface coats on human lymphocytes: freeze-drying and staining with cations.

Surface coats can be demonstrated on human peripheral blood lymphocytes by staining with ruthenium red, alcian blue, Thorotrast, and cationized ferritin, which are similar in distribution to a 40- to 65-nm layer of amorphous extracellular material recently reported on fixed, freeze-dried lymphocytes. Several additional lines of evidence, including X-ray microanalysis, suggest that the latter is not a contaminant added by freeze-drying. Freeze-drying may provide the means for a morphological assessment of the lymphocyte surface, including the extracellular coat, which may give additional insight into the immune response.

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.

Cellular associations in the rat oocyte‐cumulus cell complex: Morphology and ovulatory changes

AbstractCumulus‐oocyte complexes were isolated from the follicles of proestrous rats or from the oviducal ampullae of estrous rats. The zona pellucida of some complexes was dissolved before fixation. The follicular cumulus cells were seen to be held together mainly by long processes, which often extended over a distance of several cells. Large numbers of straight processes from the corona radiata cells, passing to the oocyte, surface, were seen in the space formerly occupied by the zona pellucida. Oocyte microvilli were uniformly short; none traversed the zona.The postovulatory complexes were covered by amorphous extracellular material which also filled the spaces between the cells. By lysis of this material with hyaluronidase the cumulus cells were detached. The surfaces of these cells were covered with blebs.By testing the ability of hyaluronidase to remove the corona cells from the zona pellucida of complexes isolated around the time of ovulation, it was found that the completion of retraction of the corona cells processes occurred in the oviduct, immediately after ovulation. It is suggested that the oviducal environment may influence the final step of the withdrawal of the corona cells' projections from the zona pellucida.