Search PubMed⌕ Search

Biomedical subjects

E M Eddy

Publications and source records attributed to E M Eddy.

At least 73 records · Page 4Linked to original sources
PubMed1989

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells.

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.

Adenosine Triphosphate↗
PubMed1989

Lactotransferrin gene expression in the mouse uterus and mammary gland.

The mouse mammary gland and uterus expressed the gene for the secretory protein lactotransferrin under various physiological conditions. Lactotransferrin, however, was induced by estrogen in a time- and dose-dependent fashion in the uterus of the immature mouse, but was not affected by estrogen in the mammary gland. Differences were also found in the expression of lactotransferrin in mammary glands and uteri of adult females during lactation. A high level of the protein was detected by immunocytochemistry in uterine epithelial cells 1 day after parturition, but immunoreactivity disappeared quickly thereafter. Lactotransferrin message was, however, relatively abundant in the mammary gland at the end of the lactation period. The presence of lactotransferrin in various tissues also was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Two forms of immunoreactive material was detected by this method; a 70K band was found in uterine luminal fluid from the estrogen-stimulated immature mouse and in homogenates of lung, vagina, mammary gland, oviduct, spleen, lymph node, and uterus of the adult female mouse, and a 65K band was detected in submaxillary gland, kidney, ovary, and all of the above tissues. Brain and duodenum had no detectable immunoreactive material. A transient appearance of lactotransferrin was observed in the uterine luminal fluid of pseudopregnant mice. These changes in the level of lactotransferrin in the uterus and mammary gland under various physiological conditions suggest that the regulation of this protein's expression is tissue specific.

Animals↗
PubMed1989

Receptor-mediated endocytosis and differential synthesis of mannose 6-phosphate receptors in isolated spermatogenic and sertoli cells.

Mannose 6-phosphate (Man 6-P) receptors participate in the targeting of both newly synthesized and extracellular acid hydrolases to lysosomes, and also may mediate the effects of a number of growth factors. In this study, Man 6-P receptors were isolated from [35S]methionine-labeled germ cells and Sertoli cells by phosphomannan-Sepharose affinity chromatography and were analyzed by polyacrylamide gel electrophoresis and autoradiography. Mouse pachytene spermatocytes and round spermatids isolated by unit gravity sedimentation synthesized predominantly the 46,000 mol wt (Mr) cation-dependent Man 6-P receptor and only low levels of the 270,000 Mr cation-independent Man 6-P receptor. In contrast, Sertoli cells synthesized substantial amounts of the cation-independent Man 6-P receptor, but little of the cation-dependent receptor. To determine if these receptors function on the cell surface, we have monitored Man 6-P receptor-mediated endocytosis in isolated germ cells and Sertoli cells. In pachytene spermatocytes and round spermatids, [125I]Man 6-P-bearing ligands were internalized and processed to lower Mr forms by an endocytic mechanism that was time dependent, proportional to cell number, and inhibited by Man 6-P. Like germ cells, Sertoli cells in primary culture endocytosed radiolabeled Man 6-P-bearing ligands at levels that were about 10% of the endocytic activity measured for 3T3 fibroblasts. This low endocytic activity correlates with the levels of the cation-independent Man 6-P receptor synthesized by germ cells, but not with the higher levels synthesized by Sertoli cells. Membrane binding assays verified the high steady state levels of the cation-independent Man 6-P receptor in Sertoli cells, suggesting that the low endocytic activity detected in these cells may result from restricted expression of the cation-independent receptor on the cell surface. These results indicate that both spermatogenic and Sertoli cells have surface Man P-6 receptors capable of mediating endocytosis. However, these cells exhibit marked differences in the expression of the cation-independent and -dependent Man 6-P receptors, perhaps reflecting differential roles of these receptors in protein trafficking and/or intercellular communication during germ cell differentiation.

Animals↗
PubMed1988

Identification of a protein in the fibrous sheath of the sperm flagellum.

The fibrous sheath is a unique cytoskeletal component in the principal-piece segment of the mammalian sperm flagellum. Monoclonal antibody ATC was shown by indirect immunofluorescence (IIF) to bind to the principal piece of the flagellum of permeabilized mouse, rat, and hamster sperm, but not to that region of guinea pig, rabbit, or human sperm. IIF on isolated fibrous sheaths confirmed that the antigen was present in the fibrous sheath of mouse, rat, and hamster sperm. On Western blots of mouse spermatozoa, ATC identified a relatively insoluble major antigen with an apparent molecular weight of 67,000 (Mr 67,000). Hamster sperm fibrous sheaths contain an antigen of Mr 66,000, while rat sperm fibrous sheaths contain an antigen of Mr 65,500. The antigen was first detected in late spermatids, as determined by immunohistochemical procedures on sections of mouse, rat, and hamster testis. The antigen was not detected on Western blots of mouse brain, kidney, liver, or thymus. These results indicate that ATC recognizes a protein integral to the fibrous sheath of the principal piece of sperm detected by immunohistochemistry late in spermiogenesis that is probably restricted to the male germ cell line.

Animals↗
PubMed1988

Acrosomal constituents identified with a monoclonal antibody are modified during late spermiogenesis in the mouse.

Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.

Acrosome↗
PubMed1988

Antigens recognized by monoclonal antibody to mouse acrosomal components differ in guinea pig spermatogenic cells and sperm.

Monoclonal antibody 1D4 reacts with a glycoconjugate antigen of the developing mouse spermatid acrosome until the terminal steps of spermiogenesis. Although 1D4 does not label the acrosome of mouse epididymal spermatozoa, it does bind to the acrosomal region of guinea pig epididymal sperm. Here we report that the antigens recognized in extracts of guinea pig spermatogenic cells and sperm are different from those detected in mouse spermatids. Three major bands of reactivity with apparent molecular weights (Mr) of 97,000-145,000, 180,000, and greater than 200,000 were detected in extracts of guinea pig sperm. Soluble antigens with the same molecular weights were released after the acrosome reaction was induced with ionophore A23187. On two-dimensional immunoblots, 1D4 recognized a microheterogeneous population of molecules in guinea pig sperm extracts. The molecules recognized by this antibody are not major Concanavalin A receptors and do not react strongly with periodic acid-Schiff's stain or periodic acid-dansylhydrazine. However, comparisons of immunoblots of sperm extracts indicate that 1D4 reacted with antigens having similar molecular weights to glycoconjugates recognized by antibodies J1 and C6. Immunofluorescent labeling of guinea pig germ cells showed that 1D4 reacted only with the acrosomes of developing spermatids and sperm, and occasionally with the juxtanuclear region of spermatocytes. In general, staining was associated with the periphery of the acrosome and not with the acrosomal granule. Immunoblots of extracts of guinea pig spermatocytes, round spermatids, condensing spermatids, and sperm demonstrated that the antigens change during germ cell differentiation. Thus, 1D4 can be used as a marker of the developing acrosome for studies of the synthesis, structure, and assembly of the sperm organelle.

Acrosome↗
PubMed1988

The blood group I antigen defined by monoclonal antibody C6 is a marker of early mesoderm during murine embryogenesis.

The distribution of a blood group I-associated antigen in the postimplantation mouse embryo was examined using immunohistochemical, immunofluorescence, and immunoblotting techniques. The following results were obtained: 1. The I antigen defined by monoclonal antibody C6 was largely, but not entirely, restricted to mesodermal cells and their derivatives in day-6.5 to day-10 embryos. A gradual increase in the surface expression of the I antigen was observed as mesodermal cells moved away from the primitive streak in posterior and lateral directions between the embryonic ectoderm and visceral endoderm. I expression declined in hindlimb-bud-stage embryos in a posterior-to-anterior progression, and was not observed in fetal tissues after day 11. 2. The appearance of mesodermal cells was associated with a switch in surface carbohydrate expression. Two-color labeling experiments using SSEA-1 and C6 monoclonal antibodies suggested that mesodermal cells leaving the primitive streak lose surface Lex and acquire I. This suggestion was supported by flow-cytofluorimetric analyses of single cell preparations of day-8 and day-10 embryos: cell-surface Lex expression declined markedly between days 8 and 10, while I expression increased. 3. The glycoproteins carrying Lex and I antigens were observed to change in molecular weight during early development. Lex was identified on a broad glycoprotein band with an apparent molecular weight greater than 200,000, whereas the I antigen was identified on glycoprotein bands with apparent molecular weights of 155,000 (day 8), and 140,000, 155,000, 210,000, and 250,000 (day 10). The results indicate that the I antigen is a useful marker of early mesoderm in the mouse embryo.

Animals↗
PubMed1988

A novel hsp70-like protein (P70) is present in mouse spermatogenic cells.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.

Animals↗
PubMed1988

Expression of heat shock proteins by isolated mouse spermatogenic cells.

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.

Animals↗
PubMed1987

Isolation of viable mouse primordial germ cells by antibody-directed flow sorting.

The germ line represents a cell type of unique interest in mammals because it retains complete genotypic totipotency while undergoing significant phenotypic differentiation. Analysis of the mechanism that underlies the maintenance of this totipotency requires the ability to isolate and study all stages of the germ cell lineage. The primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. It has not previously been possible to isolate PGC in sufficient numbers and purity to facilitate biochemical and/or molecular analysis. We report here that the use of a monoclonal antibody in combination with flow cytometry does permit the isolation of reasonably large and pure yields of viable mouse PGC.

Animals↗
PubMed1987

The distribution of two cell surface determinants of mouse embryonal carcinoma and early embryonic cells.

This study examines the distribution of two carbohydrate determinants recognized by monoclonal antibodies EMA-1 and EMA-6 on cells in culture, adult mouse tissues and day 0-7 embryos, and provides partial biochemical characterization of the molecules carrying the determinants for EMA-1 on embryonal carcinoma cells. Both antigens are present on primordial germ cells of day 8-day 10 mouse embryos (Hahnel and Eddy, 1986), and are expressed recurrently on pluripotent cells during earlier embryogenesis. Both antigens are also present on mouse embryonal carcinoma cells and the same set of adult tissue types. However, the distribution of the EMA-6 determinant on adult urogenital epithelia and during embryogenesis is more restricted than that of EMA-1. The determinants are detected only on lumenal surfaces of epithelial cells, suggesting that they are either not involved in processes of cell-cell or cell-substrate interaction, or are masked or altered during such interactions. Present biochemical evidence suggests that EMA-1 determinant on Nulli SCC1 cells may be on a large glycoprotein or a glycoprotein complex that is firmly attached to the cell membrane.

Animals↗
PubMed1987

The heterogeneity of isolated adult rat Leydig cells separated on Percoll density gradients: an immunological, cytochemical, and functional analysis.

Intertubular cells, isolated from adult rat testes by collagenase dispersal under conditions designed to minimize cell damage, were fractionated on Percoll density gradients. In the gradient fractions, there was a close cellular correlation between the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), determined by cytochemistry, and other Leydig cell markers (nonspecific esterase, autofluorescence, and an antigen defined by monoclonal antibody LC-1C6). As the reagents for 3 beta HSD cytochemistry are excluded by intact membranes, Leydig cells with damaged plasma membranes were identified by 3 beta HSD reactivity in suspended cell preparations, and the total number of 3 beta HSD-positive (3 beta HSD+) cells in the same preparations was determined after lysis of the cell membrane. Whole cells were differentiated from cytoplasmic fragments by counterstaining with the nuclear dye propidium iodide, and the number of intact Leydig cells in each preparation was determined subsequently by subtracting the number of damaged nucleated 3 beta HSD+ cells from the total number of nucleated 3 beta HSD+ cells. The majority of intact isolated Leydig cells were found in gradient fractions of 1.054-1.096 g/ml density. Acute (3-H) basal and hCG-stimulated testosterone production per intact Leydig cell were dependent upon the concentration of Leydig cells per assay well, indicating that there is cooperativity among Leydig cells in vitro. There was no difference in steroidogenic function among intact Leydig cells from different fractions of the above density gradient range at assay concentrations greater than 10,000 Leydig cells/well. At lower cell concentrations, Leydig cells from gradient fractions of lower density (1.054-1.064 g/ml) produced slightly less testosterone in response to hCG stimulation than Leydig cells from more dense fractions (1.070-1.096 g/ml). Prolonging the exposure of isolated cells to the dispersal conditions caused declines in the apparent buoyant density and basal testosterone and hCG-stimulated cAMP and testosterone production of all Leydig cells, without detectable changes in cell integrity. The data indicate that both the absolute steroidogenic function and the functional heterogeneity of isolated intact Leydig cells are, at least in part, dependent upon the procedures used for their isolation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Monoclonal antibodies against rat Leydig cell surface antigens.

Monoclonal antibodies (MAbs) directed against the Leydig cell surface may be used to identify this cell in testicular preparations. Collagenase-dispersed adult rat interstitial cells were fractionated on Percoll density gradients, and Leydig cell-enriched fractions were used to prepare MAbs. Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF) on isolated testicular cells and immunocytochemical localization on paraffin sections of adult testes. In density gradient fractions, immunoglobulin (Ig) M MAbs (LC-1C6 and LC-6H6) labeled the surface of cells possessing the morphological characteristics of Leydig cells. The density gradient profiles of MAb-binding activity observed by IIF and ELISA were parallel with the Leydig cell distribution as determined by [125I]-human chorionic gonadotropin (hCG) binding, testosterone response to hCG in vitro, 3 beta-hydroxysteroid dehydrogenase histochemistry and electron microscopy. The MAbs prominently labeled most interstitial cells in sections, but there was little or no labeling of connective tissue, endothelial or seminiferous tubule cells. Both MAbs recognized components of Mr 58,000 in Western blots of Leydig cell-enriched extracts. The results indicate that LC-1C6 and LC-6H6 recognize antigens on the Leydig cell surface that are not present on other isolated testicular cells from the adult rat. These MAbs are specific markers of the Leydig cell in situ and in vitro.

Animals↗
PubMed1985

Immunodissection of sperm surface modifications during epididymal maturation.

Mammalian spermatozoa undergo changes in morphology, composition, and function during transit through the epididymis. These changes correlate with acquisition by sperm of the ability to fertilize ova. It has been found that sperm from the cauda epididymidis, but not those from the caput epididymidis, are able to bind to the zona pellucida. This would imply a modification in sperm surface characteristics. Biochemical and immunological studies have demonstrated changes in sperm surface composition during epididymal maturation. These changes involve addition of epididymal secretory products to the sperm surface, loss or alteration of existing sperm surface molecules, and possibly the unmasking of preexisting molecules or epitopes. Several laboratories have studied the epididymal secretory proteins in the rat, but a consensus has not been reached on the identification, characterization, source, and sperm surface association of these proteins. Monoclonal antibodies are beginning to be used to characterize sperm surface components and sperm maturation antigens. They are proving to be valuable tools for the dissection of epididymal maturation when used in conjunction with biochemical and physiological approaches.

Animals↗
PubMed1985

A monoclonal antibody, EC-1, derived from a syngeneically multiparous mouse alters in vitro fertilization and development.

A monoclonal antibody designated 'EC-1' was derived from a fusion of myeloma cells with lymphoid tissue from a syngeneically multiparous, but otherwise unimmunized, mouse and was selected by screening for reactivity with teratocarcinoma cells. The IgM antibody binds to the cell surface of ova, zygotes, and 2-cell embryos. Binding is not detected on the 4- or 8-cell embryo but reappears on the morula and blastocyst. EC-1 binds to the trophoblast but not to the inner cell mass of in vitro attached blastocysts and the ectoplacental cone of the peri-implantation embryo. In adult tissues, EC-1 binds to the follicular cells of the ovary, the lining epithelium of the pregnant uterus, the interstitial region of the testes and to epididymal but not testicular sperm. In nongonadal tissues EC-1 binds to an epitope located in some, but not all, regions of connective tissues associated with basement membrane. The antigen detected by EC-1, as expressed on teratocarcinoma-derived cell line PYS-2, is a large glycoprotein which is sensitive to reduction. EC-1 inhibits in vitro fertilization and partially inhibits in vitro development of in vitro fertilized ova. The possible implications of EC-1 binding and activity are discussed.

Animals↗
PubMed1985

Effects of in vivo and in vitro fertilization environments on the expression of a surface antigen of the mouse sperm tail.

Sperm maturation antigen 4 (SMA 4) is a glycoprotein secreted by the mouse epididymis that binds specifically to the sperm tail. Its fate has been examined on cauda epididymidal sperm in vivo and in vitro. SMA 4 was detected by indirect immunofluorescence (IIF) on sperm flushed from uteri of mice 5.5 h after natural or artificial insemination, but not on sperm attached to cumulus cells or zonae pellucidae of eggs recovered at that time. Detectable SMA 4 declines with time in vitro, as assayed by IIF on intact sperm or by enzyme immunoassay (EIA) of detergent extracts. After 3 h in vitro, 90% or more of sperm are not positive for SMA 4 by IIF. EIA of medium in which sperm have been incubated suggests that SMA 4 is being released from the cell surface. This time-dependent loss of SMA 4 is inhibited by mouse or rat cauda epididymidal fluid, low incubation temperature, or lack of protein in the incubation medium. However, the loss does not seem to be affected by the presence of eggs, cumulus cells, or oviduct fluids. SMA 4 is not removed from the sperm by selected treatments, suggesting that it is bound to the plasma membrane by strong, noncovalent interactions.

Animals↗
PubMed1984

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies.

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.

Animals↗