Sortilin knock-down alters the expression and distribution of cathepsin D and prosaposin and up-regulates the cation-dependent mannose-6-phosphate receptor in rat epididymal cells.

The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression and distribution of cathepsin D (CatD) and prosaposin (PSAP) in a sortilin knocked down RCE-1 epididymal cell line (RCE-1 KD) in comparison with non-transfected RCE-1 cells. In RCE-1 cells, CatD was found in the perinuclear zone and co-localize with sortilin, whereas in RCE-1 KD cells, the expression, distribution and processing of the enzyme were altered. In turn, PSAP accumulated intracellularly upon sortilin knock-down and redistributed from LAMP-1-positive compartment to a perinuclear location, remaining co-localized with CatD. Interestingly, the sortilin knock-down induced CD-MPR overexpression and a redistribution of the receptor from the perinuclear zone to a dispersed cytoplasmic location, accompanied by an increased co-localization with CatD. The increase in CD-MPR could result from a compensatory response for the proper delivery of CatD to lysosomes in epididymal cells. The intracellular pathway taken by lysosomal proteins could be an approach for addressing further studies to understand the mechanism of exocytosis and therefore the role of these proteins in the epididymis.

Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis.

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.

Epididymal α-l-fucosidase and its possible role in remodelling the surface of bull spermatozoa.

The mammalian epididymis provides an appropriate environment for sperm maturation. During the epididymal transit, spermatozoa undergo biochemical and morphological changes that lead to the acquisition of the fertilizing capacity. In this study we analysed the fucosylation status of membrane glycoproteins in the spermatozoa obtained from different regions of the bull epididymis. High amounts of fucose were detected on caput spermatozoa (R.F.I. = 1010 ± 20.35), mostly located in the post-acrosome zone. A significant decrease in the fucose levels was detected toward the cauda (R.F.I. = 540.5 ± 49.93) (P < 0.05). This decrease was in line with the increased activity of α-l-fucosidase in the cauda fluid. In sperm from the cauda, the defucosylation occurred in some proteins, whereas others showed higher fucosylation rates. A significant decrease of fucose in the gametes was observed upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I. = 1.45 ± 0.08 to 1.06 ± 0.03) (P < 0.05) indicating that the α-l-fucosidase present in the epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with specific enzyme inhibitors. These results provide direct evidence that the α-l-fucosidase from epididymal fluid participates in the fucose removal from spermatozoa, as a step of sperm maturation in the bull epididymis.