Histochemistry

, Volume 97, Issue 3, pp 213–220

Distribution of insulin binding sites on Leydig cells of rat testes using insulin-coated gold particles

  • M. Kimura
  • A. Lukinius
  • J. L. E. Ericsson
  • L. Grimelius
Article

Summary

The distribution of insulin binding sites in Leydig cells dispersed with collagenase from rat testes was studied using insulin-coated gold particles as an electron opaque ligand. Using electron microscope is convenient to distinguish Leydig cells among a variety of cells in crude preparations by their ultrastructural characteristics. Leydig cells were shown to possess insulin-binding sites on their plasma membranes. Initial binding sites of insulin were located to the microvillous surfaces. Following binding, receptor-ligand complexes seemed to move to the intermicrovillous plasma membrane, then to be internalized. Two modes of the internalization were confirmed. Most of the receptor-ligand complexes on Leydig cells appeared to be internalized via large, uncoated plasma membrane invaginations, while the remainder became internalized via small pits into vesicles. The receptor-ligand complexes were subsequently transferred to large subsurface vacuoles with electron-lucent lumens believed to correspond to endosomes. The reason why IGCs on the postendosomal pathway moving toward lysosomes was also discussed.

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References

  1. Ackerman GA, Wolken KW (1981) Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal-gold insulin complex by normal human blood cells. J Histochem Cytochem 29:1137–1149Google Scholar
  2. Ascoli M (1984) Lysosomal accumulation of the hormone-receptor complex during receptor-mediated endocytosis of human choriogonadotropin. J Cell Biol 99:1242–1250Google Scholar
  3. Bergeron JJM, Rachubinski R, Searle N, Borts D, Sikstrom R, Posner BI (1980) Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography. J Histochem Cytochem 28:824–835Google Scholar
  4. Bernier M, Chatelain P, Mather JP, Saez JM (1986) Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells. J Cell Physiol 129:257–263Google Scholar
  5. Bhalla VK, Browne ES, Sohal GS (1987a) Demonstration of hCG binding sites and hCG stimulated steroidogenesis in different populations of interstitial cells. Adv Exp Biol 219:489–513Google Scholar
  6. Bhalla VK, Rajan VP, Burgett AC, Sohal GS (1987b) Interstitial cell heterogeneity in rat testes. I. Purification of collagenase-dispersed Leydig cells by unit gravity sedimentation and demonstration of binding sites for gonadotropin in light cells versus enhanced steroidogenesis in heavier cells. J Biol Chem 262:5313–5321Google Scholar
  7. Browne ES, Flasch MV, Sohal GS, Bhalla VK (1990) Gonadotropin receptor occupancy and stimulation of cAMP and testosterone by purified Leydig cells: critical dependence on cell concentration. Mol Cell Endocrinol 70:49–63Google Scholar
  8. Charreau EH, Calvo JC, Tesone M, De Souza Valle LB, Baranao JL (1978) Insulin regulation of Leydig cell luteinizing hormone receptors. J Biol Chem 253:2504–2506Google Scholar
  9. Dehejia A, Nozu K, Catt KJ, Dufau ML (1982) Purification of rat Leydig cells: functional and morphological evaluation. Ann NY Acad Sci 383:204–211Google Scholar
  10. De Mey JR (1983) The preparation of immunoglobulin gold conjugates (IGC reagents) and their use as markers for light and electron microscopic immunohistochemistry. Immunohistochemistry 3:347–372Google Scholar
  11. Fan JY, Carpentier J-L, Gorden P, Van Obberghen E, Blackett NM, Grunfeld C, Orci L (1982) Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles. Proc Natl Acad Sci USA 79:7788–7791Google Scholar
  12. Goldfine ID, Jones AL, Hradek GT, Wong KY, Mooney JS (1978) Entry of insulin into human cultured lymphocytes: electron microscope autoradiographic analysis. Science 202:760–763Google Scholar
  13. Helenius A, Mellman I, Wall D, Hubbard A (1983) Endosomes. Trends Biochem Sci 8:245–250Google Scholar
  14. Hermo L, Clermont Y, Lalli M (1985) Intracellular pathway of endocytosed tracers in Leydig cells of the rat. J Androl 6:213–224Google Scholar
  15. Hermo L, Lalli M (1988) Binding and internalization in vitro of [125I]hCG in Leydig cells of the rat. J Androl 9:1–14Google Scholar
  16. Horisberger M, Rosset J, Bauer H (1975) Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope. Experientia 31:1147–1148Google Scholar
  17. Howland BE, Zwbrowski EJ (1976) Some effects of experimentally-induced diabetes on pituitary-testicular relationships in rats. Horm Metab Res 8:465–469Google Scholar
  18. Kerr JB, Robertson DM, De Kretser DM (1985) Morphological and functional characterization of interstitial cells from mouse testes fractionated on percoll density gradients. Endocrinology 116:1030–1043Google Scholar
  19. Lin T, Haskell J, Vinson N, Terracio L (1986) Characterization of insulin and insulin-like growth factors of purified Leydig cells and their role in steroidogenesis in primary culture: A comparative study. Endocrinology 119:1641–1647Google Scholar
  20. Lukinius A, Forsbeck K, Ericsson JLE (1988) In vitro studies on the biostability of a transferrin/gold complex. J Ultrastruct Mol Struct Res 98:325–342Google Scholar
  21. Marshall S, Garvey WT, Monzon R (1987) Shunting of insulin from a retroendocytotic pathway to a degradative pathway by sodium vanadate. J Biol Chem 262:12005–12012Google Scholar
  22. Moll UM, Thum Ch, Pfiffer EF (1986) Colloidal gold-labeled insulin complex: characterization and binding to adipocytes. Histochemistry 86:83–88Google Scholar
  23. Neutra MR, Ciechanover A, Owen LS, Lodish HF (1985) Intracellular transport of transferrin- and asialoorsomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis. J Histochem Cytochem 33:1134–1144Google Scholar
  24. Nissley SP, Haskell JF, Sasaki N, De Vroede MA, Rechler MM (1985) Insulin-like growth factors. J Cell Sci 3 [Suppl]: 39–51Google Scholar
  25. Rechler MM, Nissley SP (1985) The nature and regulation of the receptors for insulin-like growth factors. Ann Rev Physiol 47:425–442Google Scholar
  26. Rigaudiere N, Loubassou S, Grizard G, Boucher D (1988) Characterization of insulin binding and comparative action of insulin and insulin-like growth factor I on purified Leydig cells from the adult rat. International J Androl 11:165–178Google Scholar
  27. Rommerts FFG, Van Roemburg MJA, Lindh LM, Hegge JAJ, Van Der Molen HJ (1982) The effects of short-term culture and perfusion on LH-dependent steroidogenesis in isolated rat Leydig cells. J Reprod Fertil 65:289–297Google Scholar
  28. Sara VR, Hall K, Misaki M, Fryklund L, Christensen N, Wetterberg L (1983) Ontogenesis of somatomedin and insulin receptors in the human fetus. J Clin Invest 71:1084–1094Google Scholar
  29. Schlessinger J, Scheshter Y, Willingham MC, Pastan I (1980) Direct visualization of the binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci USA 75:2659–2663Google Scholar
  30. Smith RM, Jarett L (1983) Quantitative ultrastructural analysis of receptormediated insulin uptake into adipocytes. J Cell Physiol 115:199–207Google Scholar
  31. Smith RM, Cobb MH, Rosen OM, Jarett L (1985) Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors. J Cell Physiol 123:167–179Google Scholar
  32. Smith RM, Jarett L (1987) Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process. Proc Natl Acad Sci USA 84:459–463Google Scholar
  33. Smith RM, Jarett L (1988) Biology of Disease: Receptor-mediated endocytosis and intracellular processing of insulin: Ultrastructural and biochemical evidence for cell specific heterogeneity and distribution from nonhormonal ligands. Laboratory Invest 58:613–629Google Scholar
  34. Tran D, Carpentier J-L, Sawano F, Gorden P, Orci L (1987) Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway. Proc Natl Acad Sci USA 84:7957–7961Google Scholar
  35. Van Deurs B, Tonnessen TI, Petersen OW, Sandvig K, Olnes S (1986) Routing of internalized ricin and ricin conjugates to the Golgi complex. J Cell Biol 102:37–47Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • M. Kimura
    • 1
  • A. Lukinius
    • 2
  • J. L. E. Ericsson
    • 2
  • L. Grimelius
    • 2
  1. 1.Department of AnatomyAichi Medical UniversityAichiJapan
  2. 2.Department of Pathology, University of UppsalaUniversity HospitalUppsalaSweden

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