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. 1989 Feb 1;108(2):367–375. doi: 10.1083/jcb.108.2.367

Identification of rat testis galactosyl receptor using antibodies to liver asialoglycoprotein receptor: purification and localization on surfaces of spermatogenic cells and sperm

PMCID: PMC2115432  PMID: 2537315

Abstract

We have found that the rat testis contains a cell surface galactosyl receptor that is antigenically related to the minor species of rat liver asialoglycoprotein receptor (ASGP-r) and has binding affinity for galactose coupled to agarose. In immunoblotting experiments, rat testis galactosyl receptor (RTG-r) is recognized by antiserum raised against the minor ASGP-r species of rat liver (designated rat hepatic lectin- 2/3, RHL-2/3). Antiserum raised against the major species RHL-1 does not recognize an antigenic protein equivalent to RTG-r. Triton X-100- extracted rat liver and testes preparations fractionated by affinity chromatography on galactose-agarose and resolved by SDS-PAGE under reducing conditions, show that rat liver contains both the major (RHL- 1) and minor (RHL-2/3) ASGP-r species whereas rat testis displays only a receptor species comigrating with RHL-2/3. RTG-r was present throughout testicular development. The receptor was found in seminiferous tubules, cultured Sertoli and spermatogenic cells, and epididymal sperm. Indirect immunofluorescent studies show RHL-2/3-like immunoreactivity on the surface of Sertoli cell, meiotic prophase spermatocytes, spermatids, and epididymal sperm. In spermatids and sperm, the immunoreactivity is restricted to the plasma membrane overlying the dorsal portion of the head. Because of RTG-r has galactose binding affinity, is present on surfaces of Sertoli and developing meiotic and postmeiotic spermatogenic cells, and overlies a region of the intact acrosome on epididymal sperm, RTG-r may have a role in spermatogenesis and in events leading to sperm-egg recognition.

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Selected References

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  1. Abdullah M., Crowell J. A., Tres L. L., Kierszenbaum A. L. Fetuin: a serum component associated with rat Sertoli and spermatogenic cells in coculture. J Cell Physiol. 1986 Jun;127(3):463–472. doi: 10.1002/jcp.1041270317. [DOI] [PubMed] [Google Scholar]
  2. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  3. Ashwell G., Morell A. G. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol Relat Areas Mol Biol. 1974;41(0):99–128. doi: 10.1002/9780470122860.ch3. [DOI] [PubMed] [Google Scholar]
  4. Baenziger J. U., Maynard Y. Human hepatic lectin. Physiochemical properties and specificity. J Biol Chem. 1980 May 25;255(10):4607–4613. [PubMed] [Google Scholar]
  5. Bechtol K. B., Brown S. C., Kennett R. H. Recognition of differentiation antigens of spermatogenesis in the mouse by using antibodies from spleen cell-myeloma hybrids after syngeneic immunization. Proc Natl Acad Sci U S A. 1979 Jan;76(1):363–367. doi: 10.1073/pnas.76.1.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bleil J. D., Wassarman P. M. Galactose at the nonreducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein's sperm receptor activity. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6778–6782. doi: 10.1073/pnas.85.18.6778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bleil J. D., Wassarman P. M. Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell. 1980 Jul;20(3):873–882. doi: 10.1016/0092-8674(80)90334-7. [DOI] [PubMed] [Google Scholar]
  8. Breitfeld P. P., Simmons C. F., Jr, Strous G. J., Geuze H. J., Schwartz A. L. Cell biology of the asialoglycoprotein receptor system: a model of receptor-mediated endocytosis. Int Rev Cytol. 1985;97:47–95. doi: 10.1016/s0074-7696(08)62348-7. [DOI] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Chiacchia K. B., Drickamer K. Direct evidence for the transmembrane orientation of the hepatic glycoprotein receptors. J Biol Chem. 1984 Dec 25;259(24):15440–15446. [PubMed] [Google Scholar]
  11. Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev. 1972 Jan;52(1):198–236. doi: 10.1152/physrev.1972.52.1.198. [DOI] [PubMed] [Google Scholar]
  12. Drickamer K., Mamon J. F., Binns G., Leung J. O. Primary structure of the rat liver asialoglycoprotein receptor. Structural evidence for multiple polypeptide species. J Biol Chem. 1984 Jan 25;259(2):770–778. [PubMed] [Google Scholar]
  13. Feuchter F. A., Vernon R. B., Eddy E. M. Analysis of the sperm surface with monoclonal antibodies: topographically restricted antigens appearing in the epididymis. Biol Reprod. 1981 Jun;24(5):1099–1110. [PubMed] [Google Scholar]
  14. Florman H. M., Wassarman P. M. O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell. 1985 May;41(1):313–324. doi: 10.1016/0092-8674(85)90084-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fornstedt N., Porath J. Characterization studies on a new lectin found in seeds of Vicia ervilia. FEBS Lett. 1975 Sep 15;57(2):187–191. doi: 10.1016/0014-5793(75)80713-7. [DOI] [PubMed] [Google Scholar]
  16. Friend D. S., Fawcett D. W. Membrane differentiations in freeze-fractured mammalian sperm. J Cell Biol. 1974 Nov;63(2 Pt 1):641–664. doi: 10.1083/jcb.63.2.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friend D. S., Rudolf I. Acrosomal disruption in sperm. Freeze-fracture of altered membranes. J Cell Biol. 1974 Nov;63(2 Pt 1):466–479. doi: 10.1083/jcb.63.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gabel C. A., Eddy E. M., Shapiro B. M. Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components. J Cell Biol. 1979 Sep;82(3):742–754. doi: 10.1083/jcb.82.3.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Halberg D. F., Wager R. E., Farrell D. C., Hildreth J., 4th, Quesenberry M. S., Loeb J. A., Holland E. C., Drickamer K. Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. Primary structure and glycosylation heterogeneity of minor receptor forms. J Biol Chem. 1987 Jul 15;262(20):9828–9838. [PubMed] [Google Scholar]
  20. Holland E. C., Leung J. O., Drickamer K. Rat liver asialoglycoprotein receptor lacks a cleavable NH2-terminal signal sequence. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7338–7342. doi: 10.1073/pnas.81.23.7338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hubbard A. L., Wilson G., Ashwell G., Stukenbrok H. An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types. J Cell Biol. 1979 Oct;83(1):47–64. doi: 10.1083/jcb.83.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hudgin R. L., Pricer W. E., Jr, Ashwell G., Stockert R. J., Morell A. G. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem. 1974 Sep 10;249(17):5536–5543. [PubMed] [Google Scholar]
  23. Kierszenbaum A. L., Tres L. L. An automated perifusion system for the study of rat spermatogenesis in vitro. Ann N Y Acad Sci. 1987;513:146–157. doi: 10.1111/j.1749-6632.1987.tb25005.x. [DOI] [PubMed] [Google Scholar]
  24. Kierszenbaum A. L., Ueda H., Ping L., Abdullah M., Tres L. L. Antibodies to rat Sertoli cell secretory proteins recognize antigenic sites in acrosome and tail of developing spermatids and sperm. J Cell Sci. 1988 Sep;91(Pt 1):145–153. doi: 10.1242/jcs.91.1.145. [DOI] [PubMed] [Google Scholar]
  25. Koehler J. K. Studies on the distribution of antigenic sites on the surface of rabbit spermatozoa. J Cell Biol. 1975 Dec;67(3):647–659. doi: 10.1083/jcb.67.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Le Bouteiller P. P., Toullet F., Righenzi S., Voisin G. A. Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques. J Histochem Cytochem. 1979 Apr;27(4):857–866. doi: 10.1177/27.4.376693. [DOI] [PubMed] [Google Scholar]
  28. McPhaul M., Berg P. Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8863–8867. doi: 10.1073/pnas.83.23.8863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Millette C. F., Bellvé A. R. Temporal expression of membrane antigens during mouse spermatogenesis. J Cell Biol. 1977 Jul;74(1):86–97. doi: 10.1083/jcb.74.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Millette C. F., Moulding C. T. Cell surface marker proteins during mouse spermatogenesis: two-dimensional electrophoretic analysis. J Cell Sci. 1981 Apr;48:367–382. doi: 10.1242/jcs.48.1.367. [DOI] [PubMed] [Google Scholar]
  31. Monesi V. Synthetic activities during spermatogenesis in the mouse RNA and protein. Exp Cell Res. 1965 Aug;39(1):197–224. doi: 10.1016/0014-4827(65)90023-6. [DOI] [PubMed] [Google Scholar]
  32. O'Rand M. G., Irons G. P., Porter J. P. Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg. Biol Reprod. 1984 Apr;30(3):721–729. doi: 10.1095/biolreprod30.3.721. [DOI] [PubMed] [Google Scholar]
  33. O'Rand M. G., Romrell L. J. Appearance of cell surface auto- and isoantigens during spermatogenesis in the rabbit. Dev Biol. 1977 Feb;55(2):347–358. doi: 10.1016/0012-1606(77)90178-6. [DOI] [PubMed] [Google Scholar]
  34. Pierce M., Turley E. A., Roth S. Cell surface glycosyltransferase activities. Int Rev Cytol. 1980;65:1–47. doi: 10.1016/s0074-7696(08)61958-0. [DOI] [PubMed] [Google Scholar]
  35. Scully N. F., Shaper J. H., Shur B. D. Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation. Dev Biol. 1987 Nov;124(1):111–124. doi: 10.1016/0012-1606(87)90464-7. [DOI] [PubMed] [Google Scholar]
  36. Shur B. D., Hall N. G. A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida. J Cell Biol. 1982 Nov;95(2 Pt 1):574–579. doi: 10.1083/jcb.95.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tanabe T., Pricer W. E., Jr, Ashwell G. Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J Biol Chem. 1979 Feb 25;254(4):1038–1043. [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tres L. L., Kierszenbaum A. L. Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3377–3381. doi: 10.1073/pnas.80.11.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tres L. L., Smith E. P., Van Wyk J. J., Kierszenbaum A. L. Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures. Exp Cell Res. 1986 Jan;162(1):33–50. doi: 10.1016/0014-4827(86)90424-6. [DOI] [PubMed] [Google Scholar]
  41. Tung K. S., Han L. P., Evan A. P. Differentiation autoantigen of testicular cells and spermatozoa in the guinea pig. Dev Biol. 1979 Jan;68(1):224–238. doi: 10.1016/0012-1606(79)90255-0. [DOI] [PubMed] [Google Scholar]
  42. Tung P. S., Fritz I. B. Specific surface antigens on rat pachytene spermatocytes and successive classes of germinal cells. Dev Biol. 1978 Jun;64(2):297–315. doi: 10.1016/0012-1606(78)90080-5. [DOI] [PubMed] [Google Scholar]
  43. Wall D. A., Hubbard A. L. Galactose-specific recognition system of mammalian liver: receptor distribution on the hepatocyte cell surface. J Cell Biol. 1981 Sep;90(3):687–696. doi: 10.1083/jcb.90.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wassarman P. M. Early events in mammalian fertilization. Annu Rev Cell Biol. 1987;3:109–142. doi: 10.1146/annurev.cb.03.110187.000545. [DOI] [PubMed] [Google Scholar]
  45. Weigel P. H. Rat hepatocytes bind to synthetic galactoside surfaces via a patch of asialoglycoprotein receptors. J Cell Biol. 1980 Dec;87(3 Pt 1):855–861. doi: 10.1083/jcb.87.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wolf D. E., Hagopian S. S., Lewis R. G., Voglmayr J. K., Fairbanks G. Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane. J Cell Biol. 1986 May;102(5):1826–1831. doi: 10.1083/jcb.102.5.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zeitlin P. L., Hubbard A. L. Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures. J Cell Biol. 1982 Mar;92(3):634–647. doi: 10.1083/jcb.92.3.634. [DOI] [PMC free article] [PubMed] [Google Scholar]

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