Development of secretory activity in the long hyaline gland of the male migratory grasshopper,Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae)

Changes in the ultrastructure of epithelial cells from long hyaline glands of male Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae) have been examined during sexual maturation and after allatectomy. In newly emerged males, the long hyaline gland epithelium is composed of 1–3 cell layers. The cells contain almost no rough endoplasmic reticulum, inconspicuous Golgi complexes, and large numbers of free ribosomes and polysomes. Within 24 hr, the cells undergo considerable reorganization to form a 1-cell-thick layer. Changes in cytostructure include proliferation of the rough endoplasmic reticulum and the development of several elaborate Golgi complexes. The developing lumen contains a coarse fibrous material. By 3 days postemergence, columnar epithelial cells are clearly capable of considerable synthesis and export of secretory protein. Rough endoplasmic reticulum, and large, elaborate Golgi complexes are the major structural features of the cytoplasm. From day 3 to sexual maturity (day 7), no major ultrastructural changes occur, although massive accumulation of secretion in the lumen causes the epithelium to become cuboidal or flattened. Isoelectric focusing of soluble proteins from long hyaline gland secretions shows that maturing glands contain increasing numbers and quantities of secretory proteins.Allatectomy has minor effects on long hyaline gland ultrastructure. A reduction in the density of rough endoplasmic reticulum and ribosomes suggests that glands from operated males are metabolically less active. This is confirmed by qualitative and quantitative changes in the amount of secretion as revealed by isoelectric focusing. The observations are discussed in terms of the juvenile hormone control of long hyaline gland maturation.     

Development of the hyaline layer around the planktonic embryos and larvae of the asteroid Patiriella calcar and the presence of associated bacteria

Summary

The hyaline layer (HL) around the embryos and larvae of Patiriella calcar is examined by transmission electron microscopy. P. calcar hatches at the gastrula stage and develops through a lecithotrophic planktonic brachiolaria. The hyaline layer of unhatched P. calcar is poorly developed and is comprised of wispy fibrils scattered among the epithelial microvilli. Fibrils are also occasionally seen associated with the inner surface of the fertilization envelope. By the hatched gastrula stage, the hyaline layer is organized into three strata: the intervillous layer, the supporting layer and the coarse outer meshwork layer. Seven-day-old brachiolaria also have a hyaline layer comprised of three strata. In these larvae the supporting layer elevates away from the epithelial surface due to the tuft-like organization of the underlying microvilli. This results in the formation of local outpockets giving the surface of the HL a lobed appearance. Bacteria are occassionally seen in the intervillous layer, particularly in association with the outpockets. These bacteria are phagocytosed by the epithelial cells and, in larvae that have bacteria, may play an augmentive role in larval nutrition. The structure of the hyaline layer of P. calcar is compared with that of the hyaline layer of other Patiriella species to determine if it is more similar to the external coats around its planktonic (P. regularis) or benthic (P. exigua) developing congeners. The comparison shows that the hyaline layer of P. calcar is virtually identical to that of P. regularis, a similarity that may reflect the pelagic life histories of these species.     

Electron microscopy of human articular cartilage]

Scanning and transmission microscopy of the articular cartilage was performed in femoral condyles of persons at the age of 30-50 years. It was demonstrated that hyaline cartilage is covered with a protective fibrillar layer consisting of tightly pressed collagenous fibrillae with an underlying layer of fibroblastic cells. In the intracellular substance of the hyaline cartilage fibrillar structures form a complex reticular web with vertical arrangement of the main collagenous fasiculi. In the superficial layer of the hyaline cartilage the collagenous fibrillae and their fasciculi form arcade-like structures. Lacunar chondrocytes have a rough villose surface, cellular secrete is discharged as round granules through cytoplasmic membrane. Ultrastructural changes in chondrocytes are observed simultaneously with their degenerative-dystrophic changes.     

Polypeptide composition and organization of the sea urchin extraembryonic hyaline layer.

The protein composition and organization of the sea urchin extraembryonic hyaline layer was examined. Hyalin and a polypeptide of 45 kilodaltons (kDa) were present in hyaline layers isolated from 1-h-old embryos through to the pluteus larva stage. In contrast, several polypeptide species ranging in size from 175 to 32 kDa either decreased in amount or disappeared from the layer as embryonic development proceeded. Concomitant with the changes in composition, hyaline layers became progressively more refractory to dissolution by washing in Ca2+, Mg2(+)-free seawater. Incubation of intact layers, isolated from 1-h-old embryos, with proteinase K resulted in the selective digestion of hyalin and was accompanied by release of the 45-kDa polypeptide from the layers. Washing intact layers in 20 mM Tris (pH 8.0) also resulted in the selective removal of hyalin and the 45-kDa polypeptide. The Ca2(+)-precipitable protein hyalin, alone among the hyaline layer polypeptides, bound the Ca2(+)-antagonist ruthenium red. These results suggest a structural organization within the hyaline layer that is both heterogenous and dynamic throughout embryonic development.     

Lectin histochemistry of the hyaline layer around the larvae of Patiriella species (Asteroidea) with different developmental modes.

Larvae of sea stars are surrounded by an extracellular matrix called the hyaline layer. The lectin-binding properties of this matrix were investigated in an ultrastructural study of Patiriella species having different modes of development. The planktonic bipinnaria and brachiolaria of P. regularis and the planktonic brachiolaria of P. calcar demonstrated the same labeling of the hyaline layer for three lectins: Con A, SBA, and WGA. In both species the outer coarse meshwork stained for all three lectins, whereas the intervillous layer displayed patchy labeling. In the benthic brachiolaria of P. exigua, the outer coarse meshwork displayed heavy labeling for all three lectins. The heavy labeling of the outer coarse meshwork of P. exigua compared with that of the other species suggests an increased number of lectin binding sites in the hyaline layer of this species. The similar ultrastructure and histochemistry of the hyaline layer of P. regularis and P. calcar may reflect similar requirements of their extracellular cover in their planktonic environment. Lectin labeling shows that hypertrophy of the hyaline layer of P. exigua, in particular the outer coarse meshwork, involves elaboration of the carbohydrate composition of the matrix. Modifications seen in the ultrastructure and histochemistry of the hyaline layer of P. exigua appear to be associated with the evolution of benthic development.     

A morphological study on regulation of hyaline bleb formation in early embryonic cells of Cynops pyrrhogaster

Summary Presumptive ectodermal cells isolated from the animal pole region of Cynops pyrrhogaster embryos showed active formation of hyaline blebs after the eleventh cleavage. The role of the submembranous cortical layer (SCL) in hyaline bleb formation was investigated in relation to the number of cleavages. Rhodamine-phalloidin staining and transmission electron-microscopy showed structural changes in the SCL after the eleventh cleavage. Partial breakdown of the SCL by electric pulses and deformation of the SCL by treatment with cycloheximide and nocodazole induced hyaline bleb formation before the eleventh cleavage. These results suggest that the SCL plays an important role in hyaline bleb formation after the eleventh cleavage.     

Ultrastructural aspects of the surface coatings of eggs and larvae of the starfish,Pisaster ochraceus,revealed by alcian blue

Eggs of the asteroid Pisaster ochraceus demonstrate cortical granules, a thick vitelline membrane, and a poorly stained jelly coat similar to that seen on the eggs of other echinoderms. When fixed in the presence of alcian blue the jelly coat is seen to be made up of three regions, an inner layer consisting of a meshwork of fibres, a middle layer of thicker fibres, and a dense outer layer. At fertilization the cortical granules release their contents into the potential space between the vitelline layers and a low fertilization membrane consisting of the vitelline layer and a dense component of the corticle granule is formed. Initially the remaining contents of the corticle granules form an amorphous hyaline layer that fills the space between the plasma membrane and the fertilization membrane. At hatching a distinct hyaline layer is present. It persists at least to the bipinnaria stage and consists of four distinct layers. A similar layer is also located over much of the early embryonic endoderm but is lost from the regions involved in the formation of the mesenchyme cells, coelom, and mouth just before these events take place. Numerous large clear vesicles are located in the apex of all cells associated with a hyaline layer. Where the hyaline layer is lacking, only scattered vesicles are present suggesting that the vesicles may be involved in maintenance of the layer. Attempts to identify elements of the hyaline layer by immunofluorescence demonstrated that it appears to bind both antisera and control sera in a nonspecific manner.     

Structure of the extraembryonic matrices around the benthic embryos of Patiriella exigua (Asteroidea) and their roles in benthic development: Comparison with the planktonic embryos of Patiriella regularis

The extracellular matrices (ECMs) surrounding the benthic embryos and larvae of the seastar Patiriella exigua and the planktonic embryos of Patiriella regularis were examined by transmission and scanning electron microscopy. Three ECMs surround unhatched embryos: An outer jelly coat, a fertilization envelope, and an inner hyaline layer. The ECMs of P. exigua are modified for supporting benthic development. The dense jelly coat attaches the embryo to the substratum, and the fertilization envelope forms a though protective case. In comparison, P. regularis has a less dense jelly coat and a thinner fertilization envelope. The hyaline layer of both species is comprised of three main regions: An intervillous layer overlying the epithelium, a supporting layer, and a coarse meshwork layer. Unhatched P. exigua have an additional outer amorphous layer that adheres to the fertilization envelope. As a result, the hyaline layer forms a continuous ECM that unites the embryonic surface with the fertilization envelope. Embryos of P. exigua removed from their fertilization envelopes lack the outer amorphous region, have a poorly developed hyaline layer, and do not develop beyond gastrulation. It appears that the substantial hyaline layer of P. exigua and its attachment to the fertilization envelope are essential for early development and that this ECM may function as a gelatinous cushioning layer around the benthic embryos. At hatching, the amorphous layer is discarded with the envelope. In contrast, an amorphous layer is absent from the hyaline layer of P. regularis. The demembranated embryos of this species have an ECM similar to that of controls and develop normally to the larval stage. © 1995 Wiley-Liss, Inc.     

Proteolytic processing of a sea urchin, ECM-localized protein into lower mol mass species possessing collagen-cleavage activity

The hyaline layer is an apically located extraembryonic matrix, which blankets the sea urchin embryo. Using gelatin substrate gel zymography, we have identified a number of gelatin-cleaving activities within the hyaline layer and defined a precursor-product processing pathway which leads to the appearance of 40- and 38-kDa activities coincident with the loss of a 50-kDa species. Proteolytic processing of the precursor required the presence of both CaCl2 and NaCl at concentrations similar to those found in sea water. The cleavage activities utilized both sea urchin and rat tail tendon gelatins as substrates but demonstrated a species-specific cleavage activity towards sea urchin collagen. The gelatin-cleaving activities were refractory to inhibition by 1,10-phenanthroline but were inhibited by benzamidine. This latter result defines the serine protease nature of the cleavage activities. Both the 40- and 38-kDa activities were found to comigrate with gelatin-cleaving activities present in the sea urchin embryo.     

Contact-independent polarization of the cell surface and cortex of free sea urchin blastomeres

In a normal, intact sea urchin embryo blastomeres are structurally polarized so that all microvilli and cortical "pigment granules" are situated at the apical surfaces facing the hyaline layer and are absent from basolateral surfaces facing adjacent blastomeres and the internal embryonic cavity. To test the roles of intercellular contacts and the hyaline layer in the process of establishing this blastomere polarity, these two factors were experimentally eliminated; sea urchin eggs of four species were denuded of the nascent hyaline layer soon after fertilization and then cultured in calcium-free artificial seawater to prevent subsequent intercellular adhesion and contact. Such free blastomeres divided normally and still developed polarized distributions of microvilli and pigment granules resembling those of the corresponding blastomeres in intact embryos. These results indicate that the process of polarization is intrinsic to individual blastomeres (self-polarization) and that neither intercellular contacts nor adhesion of microvilli to the hyaline layer is necessary. The precise temporal and spatial coincidence of the patterns of polarization and the division cycles further suggests that a mechanistic link is maintained among cell division, blastomere polarization, and probably also a heritable component of the animal-vegetal axis.     

Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus)

Summary Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.     

The interphalangeal area of Rana pipiens: a light and electron microscopic study of the development of a fibrocartilaginous joint (symphysis)

The development of the distal interphalangeal joint in Rana pipiens hind limb was studied by light and electron microscopy. The joint was found to be a symphysis since the two articular surfaces originally capped by hyaline cartilage were separated by a joint area filled with fibrous connective tissue which ultimately was replaced by fibrocartilage. Ultrastructural studies demonstrated that the joint area development was divided into three phases. Phase I was concerned with the undifferentiated mesenchymal cells, phase II with fibroblastic and chondroblastic development, and phase III with the appearance of fibrocartilage. Changes in the cytoplasmic organelles of fibroblasts and chondroblasts, surrounding extracellular matrix, and factors related to extracellular matrix formation were described and discussed.     

Structure of the accessory reproductive glands of the male migratory grasshopper,Melanoplus sanguinipes

The accessory reproductive glands of Melanoplus sanguinipes comprise two bilateral masses of 16 tubules each, distinguishable in sexually mature insects as four white, ten short hyaline, one long hyaline, and a seminal vesicle. Over most of its length, the wall of each tubule consists of a simple glandular epithelium resting on a basal lamina, surrounded by a thin layer of circular muscle. However, near the junction with the ejaculatory duct, the wall of each tubule has a much thickened circular muscle layer and squamous or cuboidal epithelium, the region serving to regulate movement of secretion into the ejaculatory duct. Interdigitation of adjacent epithelial cells is common, and several kinds of specialized junctions occur. In the glandular region, all epithelial cells appear the same and may be flattened, cuboidal, or columnar depending on the tubule type. Except for those of the seminal vesicle, the glandular epithelial cells share ultrastructural features typical of cells engaged in the synthesis of protein for export. Despite these general similarities, in most instances subtle differences occur in the cellular ultrastructure of the epithelia of each tubule and in the appearance of their luminal secretions, suggesting that the tubules are functionally specialized.     

Hyalin release during normal sea urchin development and its replacement after removal at fertilization

All stages of sea urchin embryos through pluteus can be dissociated to their component cells through the use of a 1 M solution of glycine containing EDTA, and a similar glycine solution can be used to prevent the formation of the fertilization membrane and remove the hyaline layer material released at fertilization. The protein hyalin, which makes up the bulk of the hyaline layer, can be recovered from these glycine solutions by calcium addition and quantitative agreement was found between the hyalin released at fertilization and the hyalin present at all later developmental stages. However, embryos stripped of their hyalin at fertilization often develop normally, which is unexpected in view of the apparent involvement of the hyaline layer in developmental mechanics. Such embryos are found to have regenerated an appreciable fraction of the hyalin removed at fertilization and this regeneration occurs at the time of blastulation. Thus the regeneration appears to be stimulated by hyaline layer removal at fertilization, but it does not take place until several hours later, at the time this layer has been postulated to play a role in development.     

Storage and mobilization of extracellular matrix proteins during sea urchin development

After fertilization, sea urchin embryos surround themselves with an extracellular matrix, or hyaline layer, to which cells adhere during early development. Hyalin, the major protein component of the hyaline layer has been isolated and partially characterized in several laboratories. Although other proteins are present in the hyaline layer, little is known about their origin, distribution, or functions. The present report characterizes a set of hyaline layer proteins that are secreted after fertilization from a class of vesicles that are distinct from cortical granules. The group of proteins in these vesicles were identified by a monoclonal antibody (8d11) which recognizes a carbohydrate epitope common to each of these molecules. 8d11 polypeptides range in molecular weight from 105 to 225 kDa. Oogonia and oocytes in early stages of vitellogenesis do not express the antigen. The proteins are first observed by immunofluorescence during oogenesis as a peripheral band in mid-vitellogenic oocytes. Following germinal vesicle breakdown 8d11 moves to be distributed evenly throughout the cytoplasm. The proteins are transported to the egg surface by a cytochalasin-sensitive mechanism after fertilization, and secreted predominately within the first 30 min of development. 8d11 proteins are depleted in areas of cell contact during early embryogenesis, and become concentrated on the apical surface of ectoderm cells where they are assembled into high-molecular-weight aggregates. Three of the molecules in this group may be proteins previously described as "apical lamina" proteins. These observations provide evidence of a third pathway (cortical granules and basal lamina granules being the other two) for synthesis, storage, and exocytosis of matrix proteins that are release after fertilization.     

DISTRIBUTION OF MATURATION-PROMOTING FACTOR IN STARFISH OOCYTE STRATIFIED BY CENTRIFUGATION

In starfish, cytoplasm taken from maturing oocytes is capable of inducing breakdown of the germinal vesicle and subsequent maturation when injected into immature oocytes. The cytoplasmic factor has been designated as "maturation-promoting factor" (MPF). Ooplasm was stratified by centrifugation of maturing oocytes in density-graded Ficoll-seawater, without disrupting the cell. Three strata were distinguished beginning with the centripetal side: oil cap, hyaline layer and yellow layer. MPF activity was shown to be localized in the hyaline layer. Electron microscopic observation revealed that the hyaline layer contains Golgi complexes, many lucent vesicles and multi-vesicular bodies as distinct organelles, but seldom contains such inclusions as the lipid droplets forming the oil cap, mitochondria, yolk and pigment granules contained in the yellow layer. Based on these observations, a possible cytoplasmic component with MPF activity is discussed.     

Localisation of Sulphated Glycoconjugates during Hyaline Layer Formation in Rat Molars by Ultrastructural Cytochemistry

In order to study the presence of sulphated glycoconjugates in the first mineralised layer juxtaposed to the root dentine (the hyaline layer), we have examined the early stages of molar root development by ultrastructural cytochemistry using Cuprolinic Blue combined with enzymatic pretreatment. Upper molars from 10 to 13 day-old Wistar rats were fixed in 2.5% glutaraldehyde containing 0.05% Cuprolinic Blue in 25 mM sodium acetate, pH 5.6, containing 0.3 M MgCl2. Some specimens were previously treated with heparitinase or chondroitinase ABC. Our results showed sulphated glycoconjugate--Cuprolinic Blue complexes that appeared as electron opaque ribbon-like deposits in the unmineralised hyaline layer. Few complexes were detected adjacent to the dentinal surface. These complexes were removed by heparitinase, indicating that they contained heparan sulphate chains. In contrast, the complexes found in unmineralised cementum and root dentine were removed by chondroitinase, indicating that they contained chondroitin or dermatan sulphate chains. The complexes decreased after the initiation of mineralisation of hyaline layer and root dentine and they were no longer present in stages of fully mineralisation. We conclude that the hyaline layer only contains sulphated glycoconjugates prior to mineralisation, and that they may play a role in the regulation of the mineralisation.     

Presynaptic terminals in hyaline cells of normal and overstimulated chick inner ears

Hyaline cells are non-sensory epithelial cells of the vibrating part of the basilar membrane of chicks; they receive an extensive efferent innervation. Although these anatomical features suggest roles in auditory transduction, very little is known about the function of these cells. One possible way to understand function is by lesion experiments. We used synapsin-specific antibodies to study changes that occur in the pattern of efferent innervation in hyaline cells after lesion of the sensory epithelium induced by acoustic overstimulation. We found only small changes in hyaline cells after such trauma. These included a small increase in size and a small decrease in density of nerve terminals on hyaline cells. This suggests that hyaline cells and their nerve terminals are less susceptible to acoustic trauma than hair cells. Using neurofilament-specific antibodies we found little or no trauma-induced change in the density of nerve fibres that cross the basilar papilla and reach the hyaline cell region. This finding suggested that trauma to the hair cells does not necessarily lead to changes in the efferent fibres that cross the papilla and extend into the hyaline cell region. Using the trauma and the morphological parameters studied here, it appears that a moderate lesion in the hair cell region in the avian inner ear does not influence the hyaline cells or their innervation.     

Developmental Expression of D-Galactoside-Binding Lectin in Sea Urchin (Anthocidaris crassispina) Eggs

The spatial and temporal expression of a sea urchin (Anthocidaris crassispina) egg lectin (SUEL) during early embryogenesis was studied using antiserum raised against SUEL. Western blotting analysis revealed the presence of SUEL in all stages so far examined, from unfertilized eggs to gastrula stage embryos. Immunofluorescence and immunoelectron microscopic observation showed that SUEL was stored in small electron-dense granules which migrated to the cortex within 10 min after fertilization. SUEL was localized in the cortical cytoplasm of the blastomere during cleavage stages and subsequently migrated to the outer surface of the embryo, including the invaginated portion of the gastrula. Immunoelectron microscopic study indicated that SUEL was deposited in the hyaline layer at least at the mid gastrula stage. Migration of SUEL to the cortex was significantly reduced by treatment with cytochalasin B, suggesting that actin filaments play an important role in this translocation. Exogenously added SUEL was adsorbed at the surface of unfertilized eggs and hatched embryos, but not to embryos with fertilization membrane. Lactose inhibited this adsorption, suggesting the presence of an endogenous glycoligand(s) specific for SUEL on the surface of unfertilized eggs and in the hyaline layer. We conclude that SUEL is secreted at a certain stage of embryogenesis and specifically adsorbed to the hyaline layer. Temporal changes in extraembryonic matrices caused by SUEL seem to play an important role in developmental morphogenesis.     

A 9.6 S protein is the third calcium-insoluble component of the sea urchin hyaline layer.

A third major, calcium-insoluble component of the sea urchin (Strongylocentrotus purpuratus) hyaline layer has been purified and physically characterized. In the absence of divalent cations, the native, soluble protein has a sedimentation coefficient of 9.6 S and a molecular weight of 4.5 +/- 0.1 x 10(5). These data indicate that this large protein assumes an elongated, nonspherical conformation in solution. Its sedimentation behavior and its mobility on nondenaturing electrophoretic gels distinguish the 9.6 S protein from the 11.6 S and 6.4 S hyalin proteins we have previously characterized. That the 6.4 S, 9.6 S, and 11.6 S proteins are the major calcium-insoluble structural components of the hyaline layer is supported by the fact that we have found them in a variety of hyalin protein fractions prepared by a number of standard approaches. All three proteins are precipitated by calcium ions, thus fitting the operational definition of hyalin. Evidence is presented that the 11.6 S protein may overlie the 9.6 S protein in the hyaline layer.