Cytochemical Techniques Research Articles

Ultrastructural and cytochemical aspects of the female gonad of Geoplana burmeisteri (Platyhelminthes, Tricladida, Terricola)

The ultrastructure of the female gonad of the land planarian Geoplana burmeisteri was investigated by means of electron microscopy and cytochemical techniques. It consists of two small germaria located ventral to the intestine and of two irregular, lateral rows of vitelline follicles, both enveloped by a tunica composed of an extracellular lamina and an inner sheath of accessory cells. Accessory cell projections completely surround developing oocytes and vitellocytes. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of the rough endoplasmic reticulum (RER) and Golgi complexes. These organelles appear to be correlated with the production of egg inclusions of medium electron density, about 1.5-1.8 microm in diameter, which remain scattered in the ooplasm of mature oocytes. On the basis of cytochemical tests demonstrating their glycoprotein composition, these inclusions were interpreted as residual yolk globules. Vitellocytes are typical secretory cells with well-developed RER and Golgi complexes that are mainly involved in the production of yolk globules and eggshell globules, respectively. Eggshell globules appear to arise from repeated coalescence of small Golgi-derived vesicles and, at an intermediate stage of maturation, show a multigranular pattern. Later, after vesicle fusion, they reach a diameter of 1.3-1.6 microm when completely mature and show a meandering/concentric pattern, as is typical of the situation seen in most Proseriata and Tricladida. The content of yolk globules is completely digested by pronase, while the content of eggshell globules is unaffected. Mature vitellocytes contain, in addition, a large quantity of glycogen and lipid droplets as further reserve material. On the basis of the ultrastructural characteristics of the female gonad described above and in relation to the current literature, we conclude that G. burmeisteri appears to be more closely related to the freshwater triclads, in particular to members of the Dugesiidae, than to the marine triclads.

Microsporogenesis inAmelanchier alnifolia: sporogenous cells, microsporocytes, and tetrads

As part of an overall program aimed at increasing our knowledge of the male reproductive system of Amelanchier alnifolia Nutt., this study documents structural and developmental changes that occur in the sporogenous cells, microsporocytes, and tetrads of microspores during microsporogenesis using general cytochemical techniques in conjunction with bright field, fluorescence, and transmission electron microscopy. The sporogenous cells are thin walled and stain positively for β-1,4-glucans, pectic acids, and cellulose, but not callose. At the microsporocyte and tetrad stages of microsporogenesis, thick walls develop and stain positively for β-1,4-glucans (hemicelluloses but not cellulose), pectic acids, and callose. Thus, the eventual release of maturing microspores from the tetrads requires the digestion of all three of these carbohydrate wall materials. Postmeiotic cytokinesis is of the simultaneous type and is initiated when Golgi vesicles aggregate simultaneously into a network of cylindrical tubules in both central and peripheral cell locations of the coenocytic tetrad. Eventually, this network fuses to form the new cell walls within the microspore tetrad.

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.

Ultrastructural localization of α-galactose-containing glycoconjugates in the rat vomeronasal organ

Binding sites of Griffonia simplicifolia I-B4 isolectin (GS-I-B4), which recognizes terminal alpha-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B4 and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal alpha-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the alpha-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal alpha-galactose.

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.

Texture of the zona pellucida of the mature pig oocyte. The mammalian egg envelope revisited.

The zona pellucida (ZP) of mature pig oocytes is believed to consist of a dense filamentous meshwork, less compact on the inner and outer faces. The uneven surface of the ZP is made of unordered and stretched fibrils surrounding deep funnels which are the openings of the radial canaliculi. The topography of the ZP surface may contribute to the initial interplay between male and female gametes. Using cytochemical techniques for transmission electron microscopy (TEM), such as tannic acid and ruthenium red treatments, we found that the ZP of pig oocytes was essentially made of bundles of fibrils distributed in concentric layers (except in the innermost and outer parts). A correlation appears between the dense structure of the core layer of the ZP and its texture: it is constituted of superposed layers of fibril bundles, whereas only a random meshwork is found in a very thin innermost and in the outer layer. The fascicular configuration may control the permeability of the ZP, giving its semi-rigidity and elasticity, and may facilitate sperm penetration. The liquid crystal-like design of the core layer of the ZP is similar to textures found in the the vitelline envelope (zona radiata) of other vertebrates and possibly of all the deuterostomes. Such texture is probably related to the unique ZP protein composition and to a coordinated synthesis.

Formation of the egg envelope of a teleost, Dicentrarchus labrax (L.): immunochemical and cytochemical detection of multiple components

The formation of the egg envelope in a teleost, Dicentrarchus labrax (L.), was analysed at histological and ultrastructural level. The sequential deposition of three main layers (Z1, Z2 and Z3) constitutes the extracellular matrix throughout oocyte development. Various findings indicate that these subunits are biochemically distinct: (1) periodate- and phosphotungstic acid-reactive carbohydrates are obviously detected only in the Z1, that constitutes the initial deposit of the egg envelope in early lipidic oocytes; (2) a monoclonal antibody (DLE7) against egg envelope polypeptides did not immunostain the Z1 and the underlying Z2; (3) the antigenic determinants recognised by DLE7, thought to be exogenous in origin (synthesised in the liver), are incorporated in the inner layer (Z3). In addition, DLE7 immunostained a thin layer, assembled together with Z3. This line has not yet been described in teleost eggs and was named Z1a. This study first describes at fine cytological level the contribution of exogenous proteins to formation of the different egg envelope layers. Results obtained with conventional, immunochemical and cytochemical techniques suggest multiple synthetic sources (exogenous and follicular) of egg envelope proteins.

Structural analysis of the Lupinus mutabilis seed, its flour, concentrate, and isolate as well as their behavior when mixed with wheat flour

A description is made of the Lupinus mutabilis seed microstructure and its morphological changes, as a result of several physical (milling) and chemical procedures (alcohol, alkaline and acid treatments) used to obtain debittered and defatted lupin flour (lf), lupin protein concentrate (lpc), and lupin protein isolate (lpi). These changes were studied via light microscopy (LM), using both topographic (papanicolaou, generally employed for animal tissue identification), and cytochemical (Schiff reagent—Blue-Black Naphtol, PAS-BBN) techniques as well as scanning electronic microscopy (SEM). These techniques were also applied to the structural analysis of lf, lpc, lpi and mixtures of wheat flour (wf) with lf, lpc, and lpi for making the corresponding doughs. The flour produced after milling the L. mutabilis seed exhibited a large number of proteinaceous bodies surrounded by a cellular wall. The chemical treatments used for L. mutabilis concentrate and isolate obtention caused evident alterations in the microscopic appearance of the cellular walls, which ranged from an apparent thinning of the surface to an almost complete disruption of the structure, indicating that the protein enrichment procedure may influence the microstructural features. The lm analyses of the wf/lf, with lpc, and lpi mixtures for making doughs, showed that the gluten matrix of the dough is less interconnected in the wf–lf dough compared to wheat flour lupin concentrate and lupin isolates doughs, respectively, probably because of some conformational changes of lupin proteins originated by chemical procedures used.

Histochemistry for the research in veterinary science: the legacy of Giuseppe Aureli

Twenty years ago, on April 24th 1984, Giuseppe Aureli, Founding Member and President of the Italian Society of Histochemistry from 1971 to 1973, Director of the Institute of Anatomy of Domestic Animals, Histology and Embryology and Dean of the School of Veterinary Medicine of the University of Milan, died suddenly and before his time. Following in the footsteps of Carlo Bignardi, he started from the time he was a student, to deal with the research approaches of Maffo Vialli, founder of histochemistry in Italy Head in Milan of a skilled group of scientists, he developed a sound and reliable research school mainly based on the use of ever more advanced histochemical and cytochemical techniques to study, in the extensive area of veterinary science, different problems aimed at developing either basic knowledge or animal production.

A Comparative Cytochemical Study of the Dufour Gland in the Eusocial Bee Apis mellifera Linne, 1758 and Melipona bicolor Lepeletier, 1836

Dufour glands of Apis mellifera and Melipona bicolor were studied under light and transmission electron microscopy, using the cytochemical techniques of mercury bromophenol blue for protein detection, imidazole-buffered osmium tetroxide selective staining of unsaturated lipids, lanthanum nitrate for intercellular junction identification and zinc-iodide-osmium tetroxide for cytoplasmic endomembrane visualization. The results in both species corroborated the lipid nature of the gland secretion and showed in A. mellifera the poverty of the synthetic machinery in the worker gland cells in comparison with the queen, as expected by previous biochemical analyses. The pathway of the exogenous compounds of the secretion is intracellular, since substances can penetrate the cell folds and intercellular junctions, but their access to the gland lumen is barred by the apical intercellular junctions.

Regular Papers / Articles OrdinairesIntine wall modifications during germination ofZygophyllum fabago(Zygophyllaceae) pollen grains

The composition of the inner layer (intine) of mature, activated, and germinated Zygophyllum fabago L. (Zygophyllaceae) pollen grains was studied. Cytochemical techniques showed neutral and acidic polysaccharides to be the major component of the thin and unlayered intine. The intine lacks lipids, with only scattered lipid globules being observed near the plasma membrane. Immunocytochemical localization of esterified and unesterified pectins in the intine was performed to determine the behaviour (permeability and elasticity) of germinal apertures. The high density of unesterified pectins in the intine of Z. fabago may be related to harmomegathic changes, which increase the elasticity of the intine during hydration and germination processes. A new layer was deposited in germinated pollen grains, recognized by 1,3-β-glucan (callose) antibodies; this layer plays a role in keeping the grains swollen during the germination process and probably forms a selective barrier to control the movement of substances through the pollen walls. Indeed, the composition of the Z. fabago intine was related to both the hydration process preceding germination and the passage of allergenic proteins through it.Key words: callose, germination, intine, pectins, pollen grains, Zygophyllum fabago.

Starch from Hull‐less Barley: Ultrastructure and Distribution of Granule‐Bound Proteins

ABSTRACTStarch granules isolated from waxy, normal, and high‐amylose hullless barley grains were examined by transmission electron microscopy with cytochemical techniques. The micrographs showed two distinct regions of different sizes: 1) densely packed granule growth rings (which varied in size and number depending on the genotype), and 2) a loose filamentous network located in the central region of the granule. The granule ring width decreased with increasing amylose content. In all three genotypes, the growth rings closer to the granule surface were narrower in width than those within the granule interior. The waxy starch had wider intercrystalline amorphous growth rings, semicrystalline growth rings, and more open crystalline lamellae than normal and high‐amylose starches. Granule bound proteins (mainly integral proteins) were located in the central and peripheral (growth ring) regions of the granule.

Localization and distribution of laminin in the basal lamina of certain mechanoreceptors—an immunogold study

The applied immunogold cytochemical technique in investigating the cytologic distribution of the laminin (LAM) molecule in the capsulated Pacinian and Herbst mechanoreceptors shows the presence of LAM around most elements of the receptor structures. The LAM immunoreactivity (LAM-IR) is best expressed in the vicinity of the perineural capsule cells of both receptor types, where it is primarily concentrated around the perinuclear regions as well as the cytoplasmic lamellae. Such a localization overlaps with the already known ultrastructural localization of a basal lamina (BL) around these cells. Laminin immunoreactivity is less well expressed around the modified Schwann cells. Even in these cells, however, there is an apparent immunoreaction around the cytoplasmic lamellae regardless of the lamellar location. In both receptor types, there is no LAM-IR in the cells of the subcapsular space. Of particular significance we consider the localization of gold particles (respectively the presence of a BL) between the innermost lamellae of the modified Schwann cells and the non-myelinated part of the receptor nerve fiber and their endings, as well as around the axoplasmic protrusions of the nerve endings. We discuss the role of the BL and LAM in the investigated rapidly adapting mechanoreceptors and their trophic influence upon the sensory regions. We also assume the arresting and selective effect of these membranes in building up the ion channels of the axolemma which probably has a certain importance in mechanotransduction.

A structural study of the retinal photoreceptor, plexiform and ganglion cell layers following exposure to UV-B and UV-C radiation in the albino rat

Over the last two decades, ultraviolet radiation levels (UV), reaching the Earth's surface, have been increasing at a rate of 1.5% per each 1% loss of the ozone layer. Moreover, artificial UV-sources have also proliferated and contributed to the rising UV-stress that many organisms have to face. To assess how the vertebrate retina responds to an exposure of short wavelength UV, we focused our attention on the rat retina, observing photoreceptor (containing outer and inner segments of rods and cones), inner plexiform, and ganglion cell layers by light and transmission electron microscopy using conventional and cytochemical techniques. We analyzed how cells of the layers in question responded to a 30 min exposure to UV-C and UV-B radiation with doses of 7200 and 590 J/cm 2, respectively. The results show that there are significant changes in the nuclei and cytoplasmic organelles of the exposed retinae when compared with those of the unexposed controls. The changes include an increase in heterochromatin, distension of rough endoplasmic reticulum, mitochondrial disruptions, and increases in the number of myelin bodies. The recorded morphological changes, especially those of the ganglion cells, are suggestive of apoptotic processes and show that the exposure of vertebrate retina to wavelengths ranging from 254 to 312 nm can produce alterations that are likely to impact negatively on the retina's proper functioning.

Identifying and characterizing the joint cavity-forming cell.

For many years, a large body of circumstantial evidence supported the notion that the synovial membrane produced the hyaluronan-rich synovial fluid. A quantitative cytochemical technique for uridine-diphospho glucose dehydrogenase (UDPGD) activity established that fibroblast-like cells on the intimal surface of the synovial lining made a specific contribution to maintaining these glycosaminoglycan levels. Our studies have aimed to determine the mechanisms that control the attainment and persistence of this differentiated phenotype, and have recently focused on their appearance during joint cavity development in the embryonic limb; a process that is dependent upon skeletal movement. These in situ micro-biochemical studies have shown that cells bordering the presumptive joint cavity exhibit raised UDPGD activity, are associated with a matrix rich in hyaluronan and show immobilization-induced loss in such characteristics. Together with complimentary studies in adult joints, this suggests that mechanical stimuli promote the acquisition of this joint line-forming phenotype. For this reason our studies have attempted to identify the 'up-stream' mechano-dependent factors that control these events. Endothelial cells respond to mechanical stimuli by activating, via phosphorylation, mitogen activated protein kinase/extracellular signal-regulated kinase (MAPkinase/ERK). Using phospho-specific anti-ERK-1/2 antibodies we have shown that immunolabelling of developing limbs shows a clear joint line-selective activation during cavitation, with little if any labelling within neighbouring elements, and that this is abolished in immobilized limbs. In an attempt to facilitate the final mechanistic deciphering of these responses we have used an in vitro-based approach and found by Western blotting that active ERK-1/2 expression was increased in cultured articular surface cells following application of dynamic mechanical strain. Intriguingly, the use of a selective inhibitor (PD98059) of ERK activation by its classical activating kinase, Mek, to restrict such strain-induced increases, produced an enhanced strain-related increase in UDPGD mRNA expression. This suggests that mechano-dependent ERK activation serves a feedback regulatory role during differentiation of these cells. Whilst it is clear that these in vitro experiments serve a useful function, it is clear that they generally take little regard of the influence that might be provided by cell-cell and cell-matrix interactions within the developing limb's complex and dynamic environment and architecture. It is therefore imperative that we attempt to bridge the gap between the cell biology of such phenomena on the one hand, and the morphological approach to this same problem on the other.

Acromial spur formation in patients with rotator cuff tears

In this study we analyzed the acromial spurs of 15 patients with impingement syndrome undergoing open rotator cuff repair. Mineral apposition analysis and quantitative cytochemical techniques for glucose-6-phosphate dehydrogenase (G6PD) activity (pentose phosphate pathway), alkaline phosphatase (ALP) activity (osteoblast activity), and tartrate-resistant acid phosphatase (TRAP) activity (osteoclast phenotype) were used to examine the distribution and level of activity of selected marker enzymes within the acromial spur insertion into the coracoacromial ligament in order to establish whether local behavior of bone cells is consistent with the proposed secondary development of the acromial spur. Our results indicate that G6PD and ALP activity was higher in osteoblasts on the inferior surface compared with the superior surface of the acromial spur in all patients ( P < .001). This area correlated to the most intense area of mineral apposition shown by dual tetracycline labeling. TRAP activity revealed a heterogeneous distribution within the samples. A greater G6PD activity per cell (mean increase of 87%) was seen at the tip compared with that in post- and pre-tip zones within the coronal plane ( P < .0002). The qualitative and quantitative enzyme analyses show that the acromial insertion of the coracoacromial ligament is actively involved in bone turnover. The spatial distribution patterns of metabolically active bone-forming osteoblastic cells compared with a heterogeneous distribution of TRAP-positive osteoclasts provide evidence of bone remodeling consistent with the morphologic contours of the acromial enthesis. The sites of oxytetracycline labeling appear to correlate with the sites of high ALP and G6PD activity, which supports the concept of spur formation being a secondary phenomenon in the presence of established rotator cuff tears.

Cytochemical characterization of eosinophilic leukocytes circulating in the blood of the turtle (Chrysemys dorbignih)

Eosinophils and neutrophils are granulocytic leukocytes that are present in the blood of most vertebrates. Studies have been performed on lower vertebrates to understand the biological roles of the cells in defense mechanisms and to establish phylogenetic studies and new experimental models. Whether these 2 cell types exist in reptiles is a matter of controversy. In the blood of turtles there are 2 types of granulocytes that exhibit eosinophilia, one of them with round cytoplasmic granules and the other with elongated cytoplasmic granules. It has been suggested that these cells may be eosinophils in different stages of maturation but they also may be distinct cell types, i.e. eosinophils and neutrophils. In the present study, we characterized the 2 types of granulocytes that are present in the blood of Chrysemys dorbignih, using cytochemical techniques. Type I eosinophils showed activity of nonspecific esterase, peroxidase activity that is resistant to KCN, and basic proteins. Type II eosinophils exhibited activity of trimetaphosphatase, alkaline phosphatase, nonspecific esterase, peroxidase that is sensitive to KCN, and basic proteins. These observations indicate the existence of 2 distinct cell types in the blood of Chrysemys dorbignih, type I and type II eosinophils, that correspond to eosinophils and heterophils (neutrophils) of mammals and other vertebrates.

Seasonal Alteration of the Cytosolic and Nuclear Ca2+ Concentrations in Overwintering Woody and Herbaceous Perennials in Relation to the Development of Dormancy and Cold Hardiness

Seasonal alteration of the cytosolic and nuclear Ca2+ concentrations of spruce (Picea engelmannii Parry) and brome grass (Bromus inermis Leyss) was investigated by the antimonate precipitation cytochemical technique. Electron microscopic (EM) observations revealed that electron-dense Ca2+ antimonate deposits, an indication of Ca2+ localization, were seen mainly in the vacuole, the cell wall and the intercellular space in samples of both species, collected on 14 July 1997. Few deposits were found in the cytosol and nuclei, showing a low resting level during summer months. On 8 Aug. 1997 following a decrease in daylength of 1 hour and 12 minutes, Ca2+ accumulation was initiated in spruce with increased cytosolic and nuclear Ca2+ deposits, but not in brome grass. On 8 Sept. 1997, Ca2+ accumulation occurred in the cytosol of brome grass. This followed a drop in ambient temperature to 12 °C. Cytosolic and nuclear Ca2+ deposits continued to increase in spruce. Controlled experiments confirmed that it was the low temperature, not shortening daylength, that triggered Ca2+ accumulation in brome grass. High cytosolic and nuclear Ca2+ concentrations lasted about three months in spruce from early August to early November. However, the high cytosolic and nuclear Ca2+ concentrations in brome grass lasted only about 20 days from early September to the end of the month. During winter and spring, both species had low resting cytosolic and nuclear Ca2+ concentrations. The relationship between the duration of the high cytosolic and nuclear Ca2+ concentrations and the status of the developed dormancy/cold hardiness is discussed in light of current findings.

Cytodifferentiation of Bergmann glia and its relationship with Purkinje cells.

The Bergmann glia is composed of unipolar protoplasmic astrocytes in the cerebellar cortex. Bergmann glial cells locate their cell bodies around Purkinje cells, and extend radial or Bergmann fibers enwrapping synapses on Purkinje cell dendrites. During development, Bergmann fibers display a tight association with migrating granule cells, from which the concept of glia-guided neuronal migration has been proposed. Thus, it is widely known that the Bergmann glia is associated with granule cells in the developing cerebellum and with Purkinje cells in the adult cerebellum. As the information on how Bergmann glial cells are related structurally and functionally with differentiating Purkinje cells is quite fragmental, this issue has been investigated using cytochemical techniques for Bergmann glial cells. This review classifies the cytodifferentiation of Bergmann glial cells into four stages, that is, radial glia, migration, transformation and protoplasmic astrocytes, and then summarizes their structural relationship with Purkinje cells at each stage. The results conclude that the cytodifferentiation of Bergmann glial cells proceeds in correlation with the migration, dendritogenesis, synaptogenesis and maturation of Purkinje cells. Furthermore, morphological and molecular plasticity of this neuroglia appears to be regulated depending on the cytodifferentiation of nearby Purkinje cells. The functional relevance of this intimate neuron-glial relationship is also discussed with reference to recent studies in cell biology, cell ablation and gene knockout.

Cell wall reformation by pollen tube protoplasts of olive ( Olea europaea L.): structural comparison with the pollen tube wall

Mature pollen grains of olive (Olea europaea L.) were germinated in vitro in Brewbaker and Kwack medium, and emerging pollen tubes were then enzymatically digested in the presence of high osmoticum. This treatment resulted in simultaneous degradation of pollen tube walls and fragmentation of their cytoplasm, giving rise to numerous protoplasts of different sizes and different numbers of nuclei. After the protoplasts had been purified, they were cultured in Murashige and Skoog medium supplemented with auxin and cytokinin. The initial steps of cell wall reformation were studied after 12 h and 24 h of culture with a series of cytochemical techniques including periodic acid-Schiff reagent and phosphotungstic acid, as well as with electron microscopy and immunocytochemical techniques using monoclonal antibodies directed against pectins and β-(1→3)-glucan (callose). Among the components of new wall in the protoplasts, callose proved to be the earliest and most abundantly secreted polysaccharide, whereas the deposition of pectins recognized by the antibody JIM7 started several hours later. Pectins that bind JIM5 antibody were not detected in this early stage of development. Cell wall components deposited by protoplasts were compared with those present in growing pollen tubes. Callose secreted by protoplasts formed a relatively thicker layer than that found in the tubes, and pectins recognized by JIM7 were highly abundant, mostly within the cytoplasm and in the apical zone of the tubes.