Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells.
Sertoli cells were isolated from prepubertal mice and cultured in serum-free medium to determine whether they secrete glycoproteins containing mannose 6-phosphate (M6P). Assays of the conditioned medium for lysosomal enzyme precursors, which typically bear the M6P recognition marker, indicated that Sertoli cells selectively secreted beta-N-acetylhexosaminidase and alpha-mannosidase, but not beta-glucuronidase or beta-galactosidase. Sertoli cells were labeled metabolically with [35S]methionine and the conditioned medium was fractionated on a cation-independent M6P receptor affinity column. Most of the secreted proteins did not bind to the column (peak A); however, approximately 10% of the radioactivity eluted as a low-affinity fraction (peak B), and 5-11% of the recovered cpm bound to the column and were eluted with 2.5 mM M6P (peak C). The radiolabeled proteins in each fraction were analyzed by one- and two-dimensional electrophoresis and fluorography. Two protein bands with molecular weights of 30,000 and 35,000 were present in peak B. Peak C contained at least ten M6P-containing glycoproteins with molecular weights between 30,000 and 135,000 and isoelectric points < 6.5. The 35,000-molecular-weight constituent prominent both in peaks B and C was identified as procathepsin L by immunoprecipitation with a specific antibody. When pachytene spermatocytes and round spermatids were cultured overnight in the presence of peak C glycoproteins radiolabeled with 125I, both germ cell types accumulated these Sertoli M6P-glycoproteins by a receptor-mediated process that was specifically inhibited by M6P. The Sertoli M6P-glycoproteins taken up by germ cells were processed to lower molecular weight forms. These results provide evidence that M6P receptors on the surface of spermatogenic cells endocytose secrete glycoproteins that are likely to be present in the seminiferous epithelium.
O'Brien
D A
DA
Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill 27599-7500.
Gabel
C A
CA
Eddy
E M
EM
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008360:Mannosephosphates / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D012670:Seminiferous Epithelium / Q000502:physiology; D012708:Sertoli Cells / Q000378:metabolism*; D013091:Spermatogenesis; D013094:Spermatozoa / Q000502:physiology*
D004792:Enzyme Precursors; D006023:Glycoproteins; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L