Isolation of plasma membranes from purified mouse spermatogenic cells

1980 ◽  
Vol 43 (1) ◽  
pp. 279-299
Author(s):  
C.F. Millette ◽  
D.A. O'Brien ◽  
C.T. Moulding
Keyword(s):  
Cell Surface ◽  
Germ Cells ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Lectin Binding ◽  
Round Spermatid ◽  
Ultrastructural Observation ◽  
Spermatogenic Cells ◽  
Cell Surface Antigens ◽  
Total Recovery

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5′-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.

scholarly journals Temporal expression of membrane antigens during mouse spermatogenesis

1977 ◽  
Vol 74 (1) ◽  
pp. 86-97 ◽  
Author(s):  
CF Millete ◽  
AR Bellve
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Germ Cells ◽  
Surface Antigens ◽  
Spermatogenic Cells ◽  
Temporal Expression ◽  
Isolated Populations ◽  
Cell Surface Antigens ◽  
Membrane Antigens ◽  
Type A Spermatogonia

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.

scholarly journals Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.

1994 ◽  
Vol 125 (4) ◽  
pp. 795-802 ◽  
Author(s):  
J L Thomas ◽  
D Holowka ◽  
B Baird ◽  
W W Webb
Keyword(s):  
Cell Surface ◽  
Large Scale ◽  
Plasma Membranes ◽  
Immunoglobulin E ◽  
Large Fraction ◽  
Surface Antigens ◽  
Specific Cell ◽  
Cell Surface Antigens ◽  
Receptor Complexes ◽  
Ige Receptors

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.

Protein–glycolipid interactions during spermatogenesis. Binding of specific germ cell proteins to sulfatoxygalactosylacylalkylglycerol, the major glycolipid of mammalian male germ cells

1985 ◽  
Vol 63 (10) ◽  
pp. 1077-1085 ◽  
Author(s):  
C. A. Lingwood
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Immune Serum ◽  
Spermatogenic Cells ◽  
Molecular Weights ◽  
Testicular Spermatozoa ◽  
Antibody Staining ◽  
A New Technique

Specific binding of membrane proteins extracted from rat spermatogenic cells to the major glycolipid of the male germ cell has been demonstrated by affinity chromatography. A new method for the production of affinity matrices, using photoactivatable heterobifunctional cross-linking agents, has been used to immobilize sulfatoxygalactosylacylalkylglycerol. Three proteins of apparent molecular weights 68 000, 34 000, and 24 000 from spermatogenic cells have been shown to selectively and reversibly bind to this affinity matrix. Antiserum raised against the major of these species (68 000) was demonstrated to be specific for this protein by immunoblotting. A new technique for the reduction of background nonspecific antibody staining using this method is described. The immune serum has been used to localize the antigen in frozen testicular sections. The protein is present in the plasma membranes of all germ cells, but the expression is elevated for testicular spermatozoa and cells in or near the basal compartment of the seminiferous epithelium. The relevance of these findings to intercellular communication and spermatogenesis is discussed.

scholarly journals A cell surface antigen of the mouse related to xenotropic MuLv defined by naturally occurring antibody and monoclonal antibody. Relation to Gix G(rada1), G(aksl2) systems of MuLV-related antigens.

1981 ◽  
Vol 154 (3) ◽  
pp. 659-675 ◽  
Author(s):  
Y Obata ◽  
E Stockert ◽  
A B DeLeo ◽  
P V O'Donnell ◽  
H W Snyder ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Surface Antigen ◽  
Biochemical Characterization ◽  
Surface Antigens ◽  
Lymphoid Cells ◽  
Cell Surface Antigen ◽  
Mouse Strains ◽  
Cell Surface Antigens ◽  
Naturally Occurring

A new cell surface antigen of the mouse related to xenotropic murine leukemia virus (MuLV) is described. The antigen, designated G(erld), is defined by cytotoxic tests with the B6-x-ray-induced ERLD and naturally occurring antibody. G(erld) is distinct from all previously defined cell surface antigens. Monoclonal antibody with the same specificity has been developed. Inbred mouse strains are classified as G(erld)+ or G(erld)- according to the presence of absence of the antigen on lymphoid cells. G(erld)+ strains differ with regard to quantitative expression of G(erld) on normal thymocytes. The emergence of G(erld)+ tumors in G(erld)- strains indicates the presence of genes coding for the antigen even in strains not normally expressing the antigen. G(erld) has the characteristic of a differentiation antigen in normal mice. In G(erld)+ strains, high levels of the antigen are found on thymocytes with lower levels being detected on cells of spleen, lymph nodes and bone marrow. No G(erld) was detected in brain or kidney or on erythrocytes. The segregation ratios for G(erld) expression on thymocytes in backcross and F2 mice of crosses between G(erld)+ (B6, 129, and B6-Gix+) and G(erld)- (BALB/c) strains suggest that G(erld) expression is controlled by a single locus in B6, by two unlinked loci in 129, and by three unlinked loci in B6-Gix+ mice. Induction of the antigen by MuLV infection of permissive cells in vitro indicates that G(erld) is closely related to xenotropic and dualtropic MuLV; all xenotropic and dualtropic MuLV tested induced the antigen, whereas the majority of ecotropic and the two amphotropic MuLV failed to do so. As dualtropic MuLV are thought to be recombinants between ecotropic and xenotropic MuLV sequences, G(erld) coding by dualtropic MuLV may signify the contribution of the xenotropic part in the recombinational event. Serological and biochemical characterization indicates that G(erld) is related to the gp 70 component of the MuLV envelope. The relation of G(erld) to the previously defined gp 70-related cell surface antigens (Gix, G(rada), and G(aksl2) is discussed, particularly with regard to their characteristics as differentiation antigens, the genetic origin of dualtropic MuLV, and the leukemogenicity of MuLV.

scholarly journals Production of a monoclonal antibody against cell-surface glycoprotein of guinea pig adrenocortical cells.

1993 ◽  
Vol 41 (2) ◽  
pp. 235-243 ◽  
Author(s):  
Y Kameda ◽  
C Hirota
Keyword(s):  
Monoclonal Antibody ◽  
Sialic Acid ◽  
Cell Surface ◽  
Guinea Pig ◽  
Plasma Membranes ◽  
Surface Glycoprotein ◽  
Zona Fasciculata ◽  
Cell Surface Antigens ◽  
Adrenocortical Cells ◽  
Cell Surface Glycoprotein

A monoclonal antibody (MAb) that reacted with the cell-surface antigens of adrenocortical cells was generated against cell suspensions from guinea pig adrenal glands. Cell-surface membranes of the adrenocortical cells in all zones, i.e., zona glomerulosa, zona fasciculata, and zona reticularis, were labeled with the antibody. Adrenal medulla remained unlabeled. Immunoelectron microscopy showed that entire plasma membranes, i.e., plasma membranes between adjacent cells and free cell-surface membranes, including sinusoidal microvilli, were immunoreactive to the antibody. Immunoblot analysis demonstrated that the antibody bound to two prominent bands at molecular weights of approximately 62,000 and 110,000. Two bands were stained with lectin-digoxigenin conjugates. The 110 KD band reacted with Datura stramonium (DSA) and Maackia amurensis (MAA) agglutinins, indicating the presence of N-acetyl-glucosamine and sialic acid-linked alpha (2-3) to galactose; the 62 KD band reacted with SNA, indicating the presence of sialic acid-linked alpha (2-6) to galactose. In adrenocortical cells, the reaction pattern of Sambucus nigra (SNA) agglutinin was similar to that of the (MAb), whereas reaction patterns of DSA and MAA were different. Both neuraminidase digestion and prior absorption of the antibody with N-acetyl-neuraminic acid completely prevented the immunolabeling of adrenocortical cells. These results indicate that the MAb mainly recognizes the 2-6 sialylated cell-surface antigen of adrenocortical cells.

scholarly journals G(RADA1): a new cell surface antigen of mouse leukemia defined by naturally occurring antibody and its relationship to murine leukemia virus.

1978 ◽  
Vol 147 (4) ◽  
pp. 1089-1105 ◽  
Author(s):  
Y Obata ◽  
E Stockert ◽  
P V O'Donnell ◽  
S Okubo ◽  
H W Snyder ◽  
...  
Keyword(s):  
Cell Surface ◽  
Solid Tumors ◽  
Biochemical Characterization ◽  
Leukemia Virus ◽  
Mouse Strains ◽  
Cell Surface Antigens ◽  
Widespread Occurrence ◽  
Normal Tissues ◽  
Naturally Occurring

A new cell surface antigenic system of the mouse, designated G(RADA1), is described. The antigen is defined by cytotoxic tests with the A strain X-ray-induced leukemia RADA1 and naturally occurring antibody from random-bred Swiss mice and can be distinguished from all other serologically detected cell surface antigens of the mouse. Absorption tests indicate that G(RADA1) is present in the normal lymphatic tissue and leukemias of mouse strains with high spontaneous leukemia-incidence, e.g., AKR, C58, and C3H/Figge. Low leukemia-incidence strains, e.g., C57BL/6, BALB/c, and A lack G(RADA1) in their normal tissues, but a proportion of leukemias and solid tumors arising in these strains are G(RADA1)+. The relation of G(RADA1) to MuLV is shown by G(RADA1) appearance after MuLV infection of permissive cells in vitro; four of five N-tropic MuLV isolates, one of four B-tropic MuLV, and none of four xenotropic MuLV induce G(RADA1). Two MCF MuLV, thought to represent recombinants between N-ecotropic and xenotropic MuLV, also induce G(RADA1). Serological and biochemical characterization indicates that G(RADA1) is a type-specific determinant of the gp70 component of certain MuLV. The presence of natural antibody to RADA1 in various mouse strains and the emergence of G(RADA1)+ leukemias and solid tumors in mice of G(RADA1)- phenotype suggest widespread occurrence of genetic information coding for this antigen.

scholarly journals Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages

1983 ◽  
Vol 96 (1) ◽  
pp. 29-36 ◽  
Author(s):  
WA Muller ◽  
RM Steinman ◽  
ZA Cohn
Keyword(s):  
Cell Surface ◽  
Plasma Membranes ◽  
Adjacent Area ◽  
Cell Surface Antigens ◽  
Membrane Flow ◽  
Two Dimensional Gel Electrophoresis ◽  
Electron Microscope Autoradiography ◽  
Vacuole Membrane ◽  
Lysosomal Ph ◽  
Immune Precipitation

In previous publications (Muller, W.A., R.M. Steinman, Z.A. Cohn. 1980, J.Cell Biol. 86:292-314), we found that the membrane of macrophage phagolysosomes could be selectively radioiodinated in living cells, The technique required phagocytosis of lactoperoxidase covalently coupled to latex spheres (LPO-latex), followed by iodination on ice with Na(125)I and hydrogen peroxide. In this paper, we use the LPO-latex system to further analyze the composition and recycling of phagocytic vacuole membrane. Three approaches were employed to examine the polypeptide composition of the phagolysosome (PL) and plasma membranes (PM). (a) The efficiency of intracellular iodination was increased by increasing lysosomal pH with chloroquine. By one-dimensional SDS PAGE, the heavily labeled chloroquine-treated PL exhibited the same labeled polypeptides as PM iodinated extracellularly with LPO-latex. (b) Iodinated PL and PM were compared by two-dimensional gel electrophoresis. No differences in the isoelectric point and molecular weight of the major iodinated species were detected. (c) Quantitative immune precipitation was performed with five specific antibodies directed against cell surface antigens. Four antibodies precipitated similar relative amounts of labeled antigen on the cell surface and endocytic vacuole. One antibody, secreted by hybridoma 2.6, detected a 21-kdalton polypeptide that was enriched sevenfold in PL membrane. This enrichment was cell surface-derived, since the amount of labeled 2.6 was increased sevenfold when iodinated PM was driven into the cell during latex uptake. Therefore, intracellular iodination primarily detects PL proteins that are identical to their PM counterparts. Additional studies employed electron microscope autoradiography to monitor the centrifugal flow of radiolabeled polypeptides from PL to PM. Cells were iodinated intralysosomally and returned to culture for only 5-10 min at 37 degrees C. Most of the cell-associated label then redistributed to the cell surface or its adjacent area. Significant movement out of the lysosome compartment occurred even at 2 degrees C and 22 degrees C. Extensive and rapid membrane flow through the secondary lysosome presumably contributes to the great similarity between PM and PL membrane polypeptides.

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