NONCILIATED CELLS OF THE RAT EFFERENT DUCTS ENDOCYTOSE TESTICULAR SULFATED GLYCOPROTEIN-1 (SGP-1) AND SYNTHESIZE SGP-1 DERIVED SAPOSINS

Sertoli cell sulfated glycoprotein-1 (SGP-1) is a heavily glycosylated and sulfated 70 kDa protein that is secreted into the lumen of the seminiferous tubule where it binds to spermatozoa. Recent light and electron microscope immunocytochemistry has suggested that the testicular SGP-1 detaches from the surface of spermatozoa in the lumen of the efferent ducts to be endocytosed within the endocytic apparatus of the epithelial nonciliated cells. The finding of SGP-1 mRNA together with anti-SGP-1 immunogold labeling of the lysosomal compartment suggest that these cells synthesize an efferent duct form of SGP-1. In the present study, a number of different experimental approaches (ligation, tunicamycin treatment and a combination of both) in combination with quantitative electron microscope immunogold labeling and Western blot analysis were performed in order to test this hypothesis. The number of gold particles and the profile area of the early (endosomes, pale multivesicular bodies) and late (dense multivesicular bodies, secondary lysosomes) endocytic apparatus were estimated in each of the experimental groups and expressed as the number of gold particles per mum2 (labeling densities). The data revealed that ligation produced a significant reduction of anti-SGP-1 immunogold labeling of the early endocytic apparatus but not of the late endocytic apparatus. Tunicamycin treatment on the other hand produced a significant reduction of immunogold labeling of both the early and late endocytic apparatus. The combination of both treatments resulted in a more effective reduction of the labeling densities of these two endocytic compartments. These results thus indicate that the nonciliated cells of the efferent ducts are involved both in the endocytosis of the Sertoli-derived SGP-1 and in the synthesis of an efferent duct form of SGP-1 that is targeted from the Golgi apparatus to secondary lysosomes after its glycosylation. In order to determine the biosynthetic pathway of SGP-1 within the efferent ducts, an I.V. injection of S-35-cysteine followed by immunoprecipitation and SDS-PAGE revealed that SGP-1 was initially biosynthesized as a 55 kDa protein. This protein appears to be post-translationally modified to a 65 kDa form after 1 hour, which preceded the appearance of the 70 kDa form, and smaller peptides of about 15 kDa characteristic of saposins after 3-4 hours. Western blot analysis of ligated efferent ducts showed an increase in the biosynthesis of the 70 kDa form of SGP-1 when compared to untreated controls, however, it has yet to be established if this protein is secreted or retained in an intracellular compartment. These results thus further substantiate our hypothesis on the endocytosis of the Sertoli-derived SGP-1 by the nonciliated cells and the synthesis of an efferent duct form of SGP-1 as well as provide evidence for the presence of 15 kDa saposins and its 65 kDa precursor in secondary lysosomes of these cells.