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.
O'Brien
D A
DA
Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Gabel
C A
CA
Rockett
D L
DL
Eddy
E M
EM
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005966:Glucuronidase / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D012708:Sertoli Cells / Q000378:metabolism*; D013087:Spermatids / Q000378:metabolism; D013090:Spermatocytes / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*
D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; C027693:mannose-6-phosphate; D005966:Glucuronidase