scholarly journals The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics.

1993 ◽  
Vol 123 (4) ◽  
pp. 809-821 ◽  
Author(s):  
R Oko ◽  
L Hermo ◽  
P T Chan ◽  
A Fazel ◽  
J J Bergeron
Keyword(s):  
Protein Transport ◽  
Subcellular Fractionation ◽  
Electron Microscopic ◽  
Cytoplasmic Droplet ◽  
Lectin Staining ◽  
Golgi Region ◽  
Unknown Significance ◽  
Epididymal Spermatozoa ◽  
Membranous Component

The cytoplasmic droplet of epididymal spermatozoa is a small localized outpouching of cytoplasm of the tail of unknown significance. EM revealed flattened saccular elements as the near exclusive membranous component of the droplet. Light and electron microscopic immunolabeling for Golgi/TGN markers showed these saccules to be reactive for antibodies to TGN38, protein affinity-purified alpha 2,6 sialyltransferase, and anti-human beta 1,4 galactosyltransferase. The saccules were isolated by subcellular fractionation and antibodies raised against this fraction immunolabeled the saccules of the droplet in situ as well as the Golgi region of somatic epithelial cells lining the epididymis. The isolated droplet fraction was enriched in galactosyltransferase and sialyltransferase activities, and endogenous glycosylation assays identified the modification of several endogenous glycopeptides. EM lectin staining in situ demonstrated galactose and N-acetyl galactosamine constituents in the saccules. Endocytic studies with cationic and anionic ferritin as well as HRP failed to identify the saccules as components of the endocytic apparatus. Epididymal spermatozoa were devoid of markers for the ER as well as the Golgi-associated coatamer protein beta-COP. It is therefore unlikely that the saccular elements of the droplet participate in vesicular protein transport. However, the identification of Golgi/TGN glycosylating activities in the saccules may be related to plasma membrane modifications which occur during epididymal sperm maturation.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

scholarly journals Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm

Open Biology ◽  
2015 ◽  
Vol 5 (8) ◽  
pp. 150080 ◽  
Author(s):  
Catherine E. Au ◽  
Louis Hermo ◽  
Elliot Byrne ◽  
Jeffrey Smirle ◽  
Ali Fazel ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Glucose Transporter ◽  
Molecular Level ◽  
Mammalian Species ◽  
Cytoskeletal Proteins ◽  
Epididymal Sperm ◽  
Quantitative Electron Microscopy ◽  
Cytoplasmic Droplet ◽  
Germ Cell Differentiation

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body.

Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

1998 ◽  
Vol 111 (10) ◽  
pp. 1405-1418 ◽  
Author(s):  
V. Traverso ◽  
J.F. Morris ◽  
R.J. Flower ◽  
J. Buckingham
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lung Adenocarcinoma ◽  
Western Blotting ◽  
Signal Sequence ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cell Fractionation ◽  
Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

The lurcher gene induces apoptotic death in cerebellar Purkinje cells

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1183-1193 ◽  
Author(s):  
D.J. Norman ◽  
L. Feng ◽  
S.S. Cheng ◽  
J. Gubbay ◽  
E. Chan ◽  
...  
Keyword(s):  
Purkinje Cells ◽  
Nuclear Dna ◽  
Neuronal Loss ◽  
Terminal Phase ◽  
Nuclear Condensation ◽  
Electron Microscopic ◽  
Apoptotic Death ◽  
Microscopic Studies ◽  
Cerebellar Purkinje Cells

In the neurologically mutant mouse strain lurcher (Lc), heterozygous animals display cell autonomous degeneration of cerebellar Purkinje cells beginning in the second postnatal week. During the course of our studies to identify the genetic lesion responsible for this disease (Norman et al., 1991), we have formulated an hypothesis suggesting that in Lc Purkinje cells homeostasis is sufficiently perturbed to lead to the activation of programmed cell death, thus resulting in neuronal loss and the consequent neurologic disease (Heintz, 1993). To address this possibility, we have examined the properties of Lc Purkinje cells as they die during the second postnatal week. Our light and electron microscopic studies demonstrate that dying Lc Purkinje cells exhibit the characteristic morphologic features of apoptosis, including nuclear condensation, axon beading and membrane blebbing. Using an in situ end-labeling method, we have also detected nicked nuclear DNA in these cells. Furthermore, we have examined the expression of the sulfated glycoprotein 2 (SGP2), whose mRNA is induced in both T-cells and prostate epithelial cells undergoing apoptotic death. We show by in situ hybridization that SGP2 is not expressed at detectable levels in normal Purkinje cells, but that its mRNA is present in Lc Purkinje cells prior to their death. Also expression of the Kv3.3b potassium channel, which marks the terminal phase of Purkinje cell differentiation, is evident in Lc Purkinje cells prior to their death. These data demonstrate that the Lc mutation induces apoptosis in cerebellar Purkinje cells following their maturation in postnatal cerebellum. Isolation of the Lc mutation and further analysis of its action in eliciting apoptosis can provide an important opportunity for understanding the etiology of neurodegenerative disease.

Intracellular diffusion gradients of O2 and ATP

1986 ◽  
Vol 250 (5) ◽  
pp. C663-C675 ◽  
Author(s):  
D. P. Jones
Keyword(s):  
Mathematical Modeling ◽  
High Flux ◽  
Concentration Gradients ◽  
Electron Microscopic ◽  
Intracellular Diffusion ◽  
Metabolite Concentrations ◽  
Major Factors ◽  
Atp Supply ◽  
Endogenous Enzymes

Endogenous enzymes with different subcellular localizations provide in situ probes to study O2 and ATP concentration at various sites within cells. Results from this approach indicate that substantial intracellular concentration gradients occur under some O2- and ATP-limited conditions. These studies, along with electron microscopic analyses and mathematical modeling, indicate that clustering and distribution of mitochondria are major factors in determining the magnitude and location of the concentration gradients. The mitochondria appear to be clustered in sites of high ATP demand to maximize ATP supply under conditions of limited production. The size of such clusters is limited by the magnitude of the O2 gradient needed to provide adequate O2 concentrations for mitochondrial function within the clusters. Thus microheterogeneity of metabolite concentrations can occur in cells without membranal compartmentation and may be important in determining the rates of various high-flux processes.

scholarly journals IgE-mediated anaphylactic degranulation of isolated human skin mast cells

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 569-578 ◽  
Author(s):  
AM Dvorak ◽  
W Massey ◽  
J Warner ◽  
S Kissell ◽  
A Kagey-Sobotka ◽  
...  
Keyword(s):  
Mast Cells ◽  
Histamine Release ◽  
Human Skin ◽  
Human Lung ◽  
Lipid Bodies ◽  
Fusion Event ◽  
Electron Microscopic ◽  
Ultrastructural Morphology ◽  
Ige Mediated

Isolated human skin mast cells (HSMC) were prepared and cultured overnight before functional and electron microscopic studies. Mast cell suspensions were examined after stimulation with anti-IgE to produce anaphylactic degranulation or examined in buffer-incubated controls. Histamine release was measured in replicate samples. Control, isolated HSMC studied by electron microscopy were well preserved and fully granulated. Although all granule patterns reported for human mast cells were found, crystal granules were the most prevalent, as is true for HSMC in situ. Individual mast cells containing both crystal and scroll granules occurred. Lipid bodies were rare, as in HSMC in situ. Control, isolated mast cells did not express granule changes associated with either piecemeal degranulation or recovery during wound healing in situ; nor were morphologic changes of anaphylactic degranulation present. Spontaneous histamine release was 0% in control samples. Anaphylactic degranulation of isolated HSMC was accompanied by 24% maximum histamine release and characteristically showed extrusion of altered, membrane-free granules through multiple pores in the plasma membrane to the exterior of the cell. Other morphologic aspects of anaphylactic degranulation, as expressed in isolated human lung mast cells, were also present. These events included granule swelling, fusion, alteration of matrix contents, degranulation channel formation, pore formation, and shedding of granules, membranes, and surface processes. The ultrastructural morphology of isolated HSMC and their IgE-mediated degranulation shows some differences from similar studies of isolated human lung mast cells and of human lung and gut mast cells in biopsy samples. These differences include crystal granules as the predominant granule pattern, minor numbers of lipid bodies, and extrusion of granules during anaphylactic degranulation as characteristic for HSMC. By contrast, isolated human lung and gut mast cells have more scroll granules and particle granules, respectively, and more lipid bodies. In isolated human lung mast cells, anaphylactic degranulation is almost exclusively an intracellular fusion event characterized by the formation of complex degranulation channels within which altered granule matrix materials solubilize. In addition to morphologic differences between mast cells of skin, lung, or gut origin, functional differences have also been reported among mast cells of these organs. The ultrastructural morphology of isolated HSMC is identical to that of skin mast cells in biopsy samples, thereby validating the usefulness of this new source of HSMC for correlative functional and morphologic studies.

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