Immunoelectron microscopic localization of hyaluronic acid-binding region and link protein epitopes in brain

The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid.     

Chondroitin sulfate proteoglycan in the extracellular matrix of the canine superior olivary nuclei

The perineuronal extracellular matrix of the canine superior olivary nuclei was examined by the histochemical method. The extracellular matrix was stained with Alcian blue (pH 1.0 and 2.5), high iron diamine and ruthenium red. The staining intensity of Alcian blue in the extracellular matrix was remarkably reduced after chondroitinase ABC digestion but not after that of heparitinase or hyaluronidase. These results indicate that the extracellular matrix consists of proteoglycans and contains the chondroitin sulfate proteoglycan.     

Light and electron microscopical immunohistochemical localization of large proteoglycans in human tooth germs at the bell stage

Summary The immunohistochemical localization of large hyaluronate-binding proteoglycans has been studied in human tooth germs at the bell stage using a monoclonal antibody, 5D5, which is derived from bovine sclera and specifically recognizes the core protein of large proteoglycans, such as versican, neurocan and brevican, but not that of aggrecan. In the early bell stage before predentine secretion, when the enamel organs consisted of the inner and outer enamel epithelia, stratum intermedium and stellate reticulum, the enamel organs were not stained by 5D5, but the dental papillae and follicles stained strongly. Concomitant with the secretion of predentine, dentine and subsequent enamel matrix, strong 5D5 immunostaining distributed over the entire cell surfaces of secretory ameloblasts was observed. The forming enamel matrix showed strong staining. While most of the inner and outer enamel epithelia and stratum intermedium lacked staining, the cervical loop region and stellate reticulum showed weak staining. Although the forming dentine and odontoblasts appeared to lack 5D5 affinity, the predentine, dental papilla and dental follicle demonstrated moderate to strong reactivity. At the ultrastructural level, specific immunoreaction by immunogold particle deposition was clearly detected over the basal lamina of presecretory ameloblasts, secretion granules of secretory ameloblasts and the forming enamel matrix. These results indicate that a marked increase in the large proteoglycan associated with secretory ameloblasts may correlate with cell differentiation and enamel matrix biosynthesis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Electron microscopic study of condylar cartilage of rat mandible stained with ruthenium red: proteoglycans and hypertrophic chondrocytes

Ultrastructure of hypertrophic chondrocytes and extracellular matrix in condylar cartilage of rat mandible was studied in conjunction with ruthenium red staining. Special care was given to the preservation of proteoglycans in the extracellular matrix. Ruthenium red-positive granules were observed in the pericellular matrix of condylar chondrocytes, and their size and number increased around the hypertrophic cells. However, these granules disappeared in the lowest hypertrophic zone, in which uncalcified cartilage matrix was also disintegrated prior to initiation of ossification. Moreover, hypertrophic chondrocytes observed at the lowest zone appeared intact in their ultrastructural features, i.e., containing numbers of lysosomes and coated vesicles in the cytoplasm facing the blood capillaries. The results strongly suggest that the lowest hypertrophic chondrocytes in rat condylar cartilage may have an active role in the degradation and resorption of the pericellular matrix, especially proteoglycans, and uncalcified matrix, which changes seem an essential step for the initiation of endochondral ossification.     

Focal cell contacts detected by ruthenium red, triton X100 and saponin in the granulosa cells of mouse ovary

Granulosa cells in growing follicles of mouse ovary, observed after treatment with ruthenium red (RR) as described by Luft (1971a, b), appeared to be covered by a continuous well-defined layer. On the contrary, treating granulosa cells with 1% Triton X100 (Vaccaro and Brody, 1981), followed by RR staining, resulted in the complete extraction of the plasma membrane coat (Triton does not affect the basement membrane and extracellular matrix proteoglycans). The use of 0.02% saponin together, with the RR stain, or 0.1% Triton X100 followed by RR staining, allows good visualization of follicular basement membrane and extracellular matrix proteoglycans without destroying cell morphology. Using this technique, we observed the extraction of the plasma membrane coat, but focal RR-stained condensations that were unaffected by saponin or 0.1% Triton X100 treatment were observed between plasma membranes of granulosa cells located around the periphery of large Graafian follicles. In some cases, RR condensations were located at the apex of plasmalemmal evaginations, in proximity to adjacent granulosa cells. Focal condensations of RR stain were never observed in secondary follicles. Present evidence suggests that focal cell contacts are mediated by transmembrane intercalated glycoproteins or proteoglycans and consequently play a role in cell adhesion. Their presence among granulosa cells of only very large Graafian follicles may be related to the maturation process of granulosa cells.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

Summary The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using (i) monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and (ii) cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.     

Ultrastructure of Kurloff body proteoglycans after high pressure freezing, cryosubstitution and postembedding staining with cuprolinic blue

Very high pressure freezing and cryosubstitutlon of Kurloffcells preserves the ultrastructural morphology of Kurloff bodies,particularly the myelin figures, as shown by embedding in epoxyresin and conventional postembedding staining. It also preservesthe Kurloff body proteoglycans as more expanded spindle-likeshapes than does fixation with formaldehyde at atmospheric pressure.But., proteoglycans were not discernible in the Kurloff bodymatrix on either unstained or conventionally stained thin sections.The Kurloff body skeleton of proteoglycans in their native expandedshape was stained with the electron-dense cationic ministaincuprolinic blue, using thin sections embedded in LR white. Themean equatorial diameter of the spindles was 20–30 nm,while the collapsed filaments produced by aldehyde fixationwere about 10–15 nm wide. The spindles were often about200–300 nm long but could be much longer, depending onthe plane of the section. Thus, high pressure freezing, freezesubstitution, embedding in LR white, and staining with cationicdyes such as phthalocyanins seems to be a convenient way ofvisualizing intracellular proteoglycans that are well preservedand in very much like their native expanded state. cuprolinic blue high pressure freezing Kurloff cell proteoglycans ultrastructure     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.     

Intracellular and extracellular localization of fibronectin in primary cultures of aortic smooth muscle cells using immunoperoxidase cytochemistry in electron microscopy

This report describes the intracellular and extracellular localization of fibronectin at the ultrastructural level in primary cultures of aortic smooth muscle cells. Fibronectin was present in all the cisternae of the rough endoplasmic reticulum except the perinuclear cisterna, in large vesicles associated with the trans side of the Golgi complex, and in single large vesicles in the cytoplasm often associated with microtubules. The extracellular microfibrils were heavily stained. In sections parallel to the plane of growth bundles of extracellular microfibrils in continuity with arrays of intracellular microfilaments were observed (fibronexus). The basement membrane around the aortic smooth muscle cells was discontinuous and diffusely stained. The results indicate that fibronectin is localized in the cytoplasmic membranous apparatus of protein synthesis, processing, and secretion. The lack of reaction product in the flat cisternae of the Golgi complex let suggest either that fibronection may not be present in significant amounts within the flattened cisternae or that the method is insufficient in detecting the glycoprotein in this subcompartment off the Golgi complex.     

Immunofluorescence studies on cartilage matrix synthesis : The synthesis of link protein, chondroitin sulfate proteoglycan monomer and type II collagen

A comparison of the synthesis and deposition of fibrous type II collagen and the constituents of chondroitin sulfate proteoglycan (CSPG) aggregates, CSPG monomer and link protein, was made for chicken sternal chondrocytes in culture, using simultaneous double immunofluorescence and lectin localization. Chondrocytes deposited only CSPG constituents--and not type II collagen--into the extracellular matrix (ECM). Intracellular precursors of CSPG monomer were localized primarily in perinuclear regions, but were observed in other cytoplasmic vesicles as well. Link protein antibodies stained the same intracellular structures, but stained the perinuclear cytoplasm less intensely. In contrast, type II procollagen was distributed in vesicles throughout the cytoplasm and was clearly absent from the distinctive, CSPG precursor-containing vesicles. Fluorescence-labelled lectins were used to further identify intracellular membrane compartments. Wheat germ agglutinin (WGA) and Ricinus lectins (which recognize carbohydrates added in the Golgi) stained the perinuclear cytoplasm, while concanavalin A (conA) (which recognizes mannose-rich oligosaccharides added co-translationally) stained vesicles throughout the rest of the cytoplasm and not the perinuclear cytoplasm. The distinctive CSPG-containing vesicles were not stained with WGA or Ricinus agglutinins. Data presented elsewhere demonstrate that the vesicles do not react with monoclonal antibodies which recognize chondroitin sulfate (CS) or keratan sulfate (KS) determinants. Thus, we conclude that the vesicles accumulate CSPG precursors which have not been modified by Golgi-mediated processes. The data indicate that matrix molecules may be segregated selectively prior to transit through the Golgi complex. The co-distribution of link protein and CSPG monomer precursors in vesicles prior to further, Golgi-mediated modification may reflect an as yet undetermined function of these vesicles in the processing or assembly of CSPG.     

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.     

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. II. Studies in developing brain

In contrast to the intracellular (cytoplasmic) localization of chondroitin sulfate proteoglycans in adult brain (Aquino, D. A., R. U. Margolis, and R. K. Margolis, 1984, J. Cell Biol. 99:940-952), immunoelectron microscopic studies in immature (7 d postnatal) rat cerebellum demonstrated almost exclusively extracellular staining in the granule cell and molecular layers. Staining was also extracellular and/or associated with plasma membranes in the region of the presumptive white matter. Axons, which are unmyelinated at this age, generally did not stain, although faint intracellular staining was present in some astrocytes. At 10 and 14 d postnatal there was a significant decrease in extracellular space and staining, and by 21 d distinct cytoplasmic staining of neurons and astrocytes appeared. This intracellular staining further increased by 33 d so as to closely resemble the pattern seen in adult brain. Analyses of the proteoglycans isolated from 7-d-old and adult brain demonstrated that they have essentially identical biochemical compositions, immunochemical reactivity, size, charge, and density. These findings indicate that the antibodies used in this study recognize the same macromolecule in both early postnatal and adult brain, and that the localization of this proteoglycan changes progressively from an extracellular to an intracellular location during brain development.     

Ultrastructural demonstration of increased sulfated proteoglycans and calcium associated with chondrocyte cytoplasmic processes and matrix vesicles in rat growth plate cartilage

A monoclonal antibody (3-B-3) to chondroitin 6-sulfated proteoglycan was used with immunoperoxidase electron microscopy to study the relationship of chondrocyte cytoplasmic processes and matrix vesicles in rat epiphyseal growth plate cartilage. Immunoperoxidase staining of the chondrocyte plasmalemma was found at all levels in the growth plate and was most prominent in the hypertrophic zone. The plasmalemma and matrix of the cytoplasmic process often demonstrated stronger reactivity than the remainder of the cell surface. Matrix vesicles showed weak to strong surface or internal reactivity. The majority of them stained very similarly to the cytoplasmic process. X-ray microanalysis of specimens processed by rapid freezing and freeze substitution confirmed that both sulfur and calcium were localized within or in close association with both the cytoplasmic process and the matrix vesicle, suggesting a chemical combination of calcium with sulfated proteoglycans at both sites. These results indicate that there is a selective increase in the concentration of membrane-associated sulfated proteoglycan and calcium in the cell process, from which matrix vesicles may be released into the extracellular matrix.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.     

Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules. A study using acid phosphatase and ruthenium red ultrastructural histochemistry

Starfish oocytes were examined before fertilization, immediately after insemination, and during the cortical reaction by means of acid phosphatase and ruthenium red ultrastructural histochemistry. Oocyte cortical granules are composed of a lamellar body and a surrounding matrix which is subdivided into dense and light portions. In unfertilized oocytes cortical granules are not stained by ruthenium red but show a weak acid phosphatase activity in the light portion of the granule matrix. Immediately after the adhesion of the spermatozoon to the oocyte jelly coat, the light matrix portion of cortical granules appears stained by ruthenium red and shows a strong acid phosphatase activity. During the cortical reaction, cortical granules are released into the perivitelline space and the lamellar body, surrounded by the stained matrix, fuses with the fertilization envelope. Our data suggest that membrane permeability changes and enzyme activation occur in the egg when the spermatozoon binds to the oocyte jelly coat.