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  • 1
    Electronic Resource
    Electronic Resource
    Surface-associated glycosyltransferase activities in rat sertoli cells in vitro (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 36 (1993), S. 195-202 
    Details
    ISSN: 1040-452X
    Keywords: Fucosyltransferase ; Galactosyltransferase ; Sertoli cell plasma membranes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20-day-old rat testes displayed FT activity with a Vmax of 12.5 pmoles/mg protein/min and a Km of 22 μM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a Vmax of 10 pmoles/mg protein/min and a Km of 18.2 μM for GDP-[14C]-L-fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum-free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle-stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a Vmax of 50 pmoles/mg protein/min and a Km of 38.5 μM, while SCPM showed GalTase activity with a Vmax of 25 pmoles/mg protein/min and a Km of 20.8 μM for UDP-[3H]-galactose. Galactosyltransferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, ∼33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells. These results suggest that cell surface glycosyltransferases may be involved in Sertoli cell function during mammalian spermatogenesis. © 1993 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080360210
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  • 2
    Electronic Resource
    Electronic Resource
    N-cadherin mediates sertoli cell-spermatogenic cell adhesion (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Developmental Dynamics 197 (1993), S. 1-13 
    Details
    ISSN: 1058-8388
    Keywords: N-cadherin ; Seminiferous tubules ; Cell-cell adhesion ; Sertoli cell ; Spermatogenic cell ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The complex topological association of Sertoli cells and spermatogenic cells in the testis suggests the existence of cell surface adhesion molecules that regulate cellular interactions within the seminiferous epithelium. The recent report of N-cadherin mRNA expression in the mouse testis implies the involvement of this known adhesion molecule in testicular cell binding. Accordingly, here we report that (1) N-cadherin is found on the surface membranes of rat spermatogenic cells and on Sertoli cells, and (2) that N-cadherin is a partial mediator of Sertoli cell-germ cell adhesion as tested in an in vitro cell-cell binding assay. Antiserum directed against the N-cadherin cell adhesion recognition sequence was used for Western blot analysis of purified plasma membranes from Sertoli cells and from spermatogenic cells. Both membrane preparations exhibited reactivity at an appropriate Mr of about 130 kDa. In addition, immunofluorescence assays demonstrated that both germ cells and Sertoli cells were labeled by anti-N-cadherin. Finally, the antiserum was included in a cytometer-assisted cell-cell binding test to determine its inhibitory ability. The antiserum consistently reduced specific testicular cell-cell adhesion by 30%-50%. This is the first demonstration that antibodies directed against the cadherin cell adhesion recognition sequence are capable of inhibiting cell-cell interactions. Pre-incubation of either rat Sertoli cells or spermatogenic cells alone was sufficient to achieve statistically significant inhibition of intercellular adhesion. We conclude, therefore, that N-cadherin is expressed by both Sertoli cells and spermatogenic cells and that N-cadherin is one of a number of regulatory molecules mediating local cellular associations in the mammalian seminiferous tubule. © 1993 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/aja.1001970102
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  • 3
    Electronic Resource
    Electronic Resource
    Radio-iodination of plasma membrane polypeptides from isolated mouse spermatogenic cells (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 317-331 
    Details
    ISSN: 0148-7280
    Keywords: spermatogenesis ; plasma membrane proteins ; cell surface iodination ; Two-dimensional electrophoresis ; mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cell surface polypeptides of mouse pachytene spermatocytes and round spermatids (steps 1-8) have been iodinated using 1,2,3,6,tetracholoro-3α, 6α-diphenylglycouril (IODOGEN). Labeled proteins have been assayed using two-dimensional polyacrylamide electrophoresis and radioautography. Purified plasma membranes, prepared from both spermatocytes and spermatids after the iodination of intact cells, exhibit 25-30 polypeptides which label reproducibly. No significant qualitative differences are noted in the labeled polypeptide map obtained from each of the purified cell types. Iodinated proteins range in molecular weight from greater than 100k daltons to approximately 40k daltons. The isoelectric points of labeled constituents range from pI 5.7 to 7.2. Three polypeptides represent the major iodinated species: p 94/5.8, p 75/5.9, and p 53/7.1. Comparison with total plasma membrane constituents assayed using Coomassie brilliant blue indicates that many of the radioactively labeled proteins are not present in quantities sufficient to allow ready detection without isotopic techniques. As a result, many of the proteins identified autoradiographically represent newly described surface components of mouse pachytene spermatocytes and round spermatids. The preparation of purified plasma membrane fractions prior to electrophoresis ensures that all iodinated species are in fact cell surface components. Furthermore, experiments designed to assess the vectorial nature of the IODOGEN-catalyzed labeling procedure suggest that most, if not all, of the iodinated species are exposed on the external side of the cell plasma membrane. Therefore, these studies have (1) identified hitherto unrecognized plasma membrane components of mouse pachytene spermatocytes and round spermatids and (2) provided the first available biochemical data concerning the molecular orientation of particular proteins in the surface membranes of developing mouse spermatogenic cells.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120040406
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  • 4
    Electronic Resource
    Electronic Resource
    Enrichment of primary pachytene spermatocytes from the human testis (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 487-498 
    Details
    ISSN: 0148-7280
    Keywords: testis ; human ; cell separation ; germ cells ; spermatocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Normal adult human testis has been separated using a combination of mechanical and enzymatic procedures to yield a suspension of viable single cells. The predominant cell types comprising this suspension are as follows: primary pachytene spermatocytes (7% of total cells), round spermatids (17%), residual bodies and condensing spermatids (31%), and Leydig cells (15%). Separated human germ cells viewed by Nomarski differential interference microscopy closely resemble mouse spermatogenic cells in relative size and appearance. Isolation of an enriched population of human pachytene spermatocytes has been achieved using unit gravity sedimentation (STA-PUT) according to protocols originally developed for murine tissue. Pachytene cells are enriched to 75% and are contaminated only with Leydig cells and binucleated spermatid symplasts. Ultrastructural examination of isolated human pachytene spermatocytes indicates that these cells, as well as isolated round spermatids, exhibit a normal in situ morphology. Spermatocytes, for example, show numerous synaptonemal complexes, nuclear pores, annulate lamellae, and dictyosome-like saccules. Round spermatids after isolation exhibit peripheral mitochondria, annulate lamellae, developing acrosomes, and other morphological features characteristic of early spermiogenesis. Therefore, enriched populations of human spermatogenic cells seem suitable for analysis using immunofluorescent, autoradiographic, or serological methods. In particular, isolated human spermatocytes should be useful for the analysis of molecular events involved in meiosis and should facilitate investigations concerning the pathophysiology of certain human infertility conditions.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120040602
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  • 5
    Electronic Resource
    Electronic Resource
    Immunochemical identification of multiple cell surface antigens appearing during specific stages of mouse spermatogenesis (1986)
    New York, NY : Wiley-Blackwell
    Gamete Research 13 (1986), S. 199-211 
    Details
    ISSN: 0148-7280
    Keywords: spermatogenesis ; cell surface antigens ; electrophoretic immunoblots ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cell surface antigens that appear in a defined temporal sequence during mouse spermatogenesis were previously detected serologically, but not identified biochemically, with four heterologous antibodies prepared against purified populations of pachytene spermatocytes (AP), round spermatids (ARS), vas deferens spermatozoa (AVDS), and mixed seminiferous cells (ASC) [Millette and Bellvé, J Cell Biol 74:86-97, 1977]. These antigens have now been identified immunochemically on nitrocellulose blots from SDS polyacrylamide gels. Three antisera (AP, ARS, and ASC) recognize a similar subset of determinants on one-dimensional immunoblots of germ cells and plasma membranes prepared from a mixed population of late spermatogenic cells. Comparisons of minor bands to reveal differences among these antisera. AVDS exhibits the least complex binding pattern. The results indicate that at least ten surface constituents appear during the pachytene stage of meiosis, coincident with a period of maximal RNA and protein synthesis [Monesi, Exp Cell Res 39:197-224, 1965]. Furthermore, two-dimensional immunoblot comparisons of plasma membranes isolated from pachytene spermatocytes and round spermatids reveal differences between surface determinants detectable at these two spermatogenic stages. For example, ASC recognizes two newly described proteins that are restricted to pachytene spermatocytes (˜ Mr 57,000, pI 6.45) and to round spermatids (˜ Mr 39,500, pI 4.85), respectively.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120130303
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  • 6
    Electronic Resource
    Electronic Resource
    Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 22 (1989), S. 321-332 
    Details
    ISSN: 0148-7280
    Keywords: glycotransferase ; plasma membrane ; spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have demonstrated previously that spermatogenic cells in the mouse testis have high levels of fucosyltransferase activity. Furthermore, a significant portion of this activity has been localized to the cell surface (Millette et al.: Cell Biology of the Testis and Epididymis, 1987). Differential expression of fucosyltransferases and their function as ecto-enzymes may be important in the processes of sperm maturation and fertilization in mammals. Accordingly, here we report the activity levels of fucosyltransferase (FT) in spermatozoa isolated from the mouse caput and cauda epididymides. Calculated on a per cell basis, spermatozoa from the caput epididymis have significantly more FT activity than do spermatozoa from the cauda epididymis (18.07 ± 2.2 pmol/million cells compared with 2.8 ± 0.09 pmol/million cells). Furthermore, caput sperm exhibit a more significant increase in FT activity when assayed in the presence of Nonidet P-40. Calculated on the basis of cell surface area, however, FT activity remains constant on the head portion of spermatozoa isolated from all portions of the male reproductive tract and from capacitated spermatozoa. Measurements of FT activity in extracts of isolated sperm tails from cells at different stages of maturation indicate a greatly diminished activity in tails from sperm in the cauda epididymis. The total sperm surface area is composed predominantly of the plasma membrane surrounding the flagellar apparatus. Therefore, our data demonstrate that FT activity is retained selectively on the different topological regions of sperm, with losses during sperm maturation in the epididymis being restricted to the tail segment. Maintenance of high levels of FT activity of the plasma membranes of the mouse sperm head raise the possibility that FT is indeed involved in some aspects of sperm-egg recognition.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120220309
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  • 7
    Electronic Resource
    Electronic Resource
    Human spermatogenic cell marker proteins detected by two-dimensional electrophoresis (1983)
    New York, NY : Wiley-Blackwell
    Gamete Research 7 (1983), S. 227-239 
    Details
    ISSN: 0148-7280
    Keywords: testis ; human ; spermatogenic cells ; two-dimensional electrophoresis ; marker proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Highly homogeneous populations of human pachytene spetmatocytes and round spermatids have been obtained from normal adult testis using unit gravity (STA-PUT) sedimentation. Contaminating Leydig cells have been removed by density centrifugation in discontinuous Percoll gradients to yield resultant germ cell purities of 90-95% for pachytene spermatocytes and 89-96% for round spermatids. The total cellular polypeptide composition of separated human germ cells has been analyzed by two-dimensional polyacrylamide gel electrophoresis to compare 1) human and mouse pachytene spermatocytes (species specificity), 2) samples of human spermatocytes obtained from different individuals (allo specificity), and 3) pachytene spermatocytes and round spermatids from the same patients (stage specificity). Mouse and human germ cells have been found to exhibit extensive homology, but identified marker proteins limited to human spermatocytes include a group of polypeptides at p45/5.9 as well as a protein at p67/5.2. Proteins unique to mouse germ cells include component p65/5.5. Comparisons between different preparations of human pachytene spermatocytes have revealed about 90% electrophoretic homology, but some striking allotypic variations have been noted including the proteins at p45/5.9. Finally, presumptive stage-specific spermatogenic cell markers have been identified including the p45/5.9 polypeptides that are present only in human spermatocytes. Although the physiological roles of particular marker proteins have not yet been determined, the present findings indicate that purified spermatogenic cell populations may be analyzed biochemically to identify constituents important in the regulation of sperm development in man.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120070304
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  • 8
    Electronic Resource
    Electronic Resource
    Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis (1989)
    New York, NY : Wiley-Blackwell
    Gamete Research 22 (1989), S. 307-319 
    Details
    ISSN: 0148-7280
    Keywords: protein methylation ; carboxylmethyltransferase ; S-adenosylmethionine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4-5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120220308
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