Glycosidases Interact Selectively With Mannose-6-Phosphate Receptors of Bull Spermatozoa.

Glycosidases may play a role in sperm maturation during epididymal transit. In this work, we describe the interaction of these enzymes with bull spermatozoa. We found that ß-galactosidase associated to spermatozoa can be released under low ionic strength conditions, whereas the interaction of N-acetyl-ß-D-glucosaminidase and ß-glucuronidase with spermatozoa appeared to be stronger. On the other hand, α-mannosidase and α-fucosidase cannot be removed from the gametes. In addition, part of N-acetyl-ß-D-glucosaminidase, ß-galactosidase, and ß-glucuronidase can also be released by mannose-6-phosphate. Taking into account these data, we explored the presence of cation-independent- and cation-dependent-mannose-6-phosphate receptors in the spermatozoa and found that cation-independent mannose-6-phosphate receptor is highly expressed in bull spermatozoa and cation-dependent-mannose-6-phosphate receptor is expressed at a lesser extent. In addition, by immunofluorescence, we observed that cation-independent-mannose-6-phosphate receptor is mostly located at the acrosomal zone, whereas cation-dependent-mannose-6-phosphate receptor presents a different distribution pattern on spermatozoa during the epididymal transit. N-acetyl-ß-D-glucosaminidase and ß-glucuronidase isolated from epididymal fluid interacted mostly with cation-independent-mannose-6-phosphate receptor, while ß-galactosidase was recognized by both receptors. We concluded that glycosidases might play different roles in bull spermatozoa and that mannos-6-phosphate receptors may act as recruiters of some enzymes. J. Cell. Biochem. 117: 2464-2472, 2016. © 2016 Wiley Periodicals, Inc.

Changes in lysosomal enzymes and mannose-6-phosphate receptors related to sexual maturation in bull epididymis.

One of the most striking features of the mammalian epididymis is the secretion of lysosomal enzymes (LE). These LE may play a role in sperm maturation. In the present study we investigated the activity and distribution of four LE (?-galactosidase (?-Gal), N-acetyl-?-D-glucosaminidase (?-NAG), ?-mannosidase (?-Man) and ?-glucuronidase (?-Glu)) in bull epididymis at two different ages (6 months and 4 years) to determine whether these enzymes vary with sexual maturity. In young, sexually immature (SI) bulls we found high LE activity in the epididymal tissue that accounts for a developed and active lysosomal apparatus. In contrast, low LE activity was measured in sexually mature (SM) bulls, and ?-NAG and ?-Gal were mostly secreted into the lumen. We also attempted to correlate LE distribution with the expression and functionality of mannose-6-phosphate receptors (MPRs), which are thought to be involved in proper delivery of LE to lysosomes. The cation-dependent MPR was highly expressed in SI bulls, with expression decreasing during adulthood, whereas the expression of the cation-independent MPR was higher in SM than SI bulls. In addition, the four enzymes recovered from the epididymal lumen interact with both MPRs at each age. We conclude that the activity and distribution of LE in bull epididymis varies with sexual maturity and that the distribution is regulated differently by the two types of MPR. These findings could provide some molecular basis for male infertility.

Effects of Heparan sulfate acetyl-CoA: Alpha-glucosaminide N-acetyltransferase (HGSNAT) inactivation on the structure and function of epithelial and immune cells of the testis and epididymis and sperm parameters in adult mice.

Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.

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.

The distribution of mannose-6-phosphate receptors changes from newborns to adults in rat liver.

The co-existence of two types of mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still not well explained. Some evidence suggests that the CI-MPR could be actively involved in the regulation of growth factors in the early stages of mammalian organ development. In this study, it was demonstrated that both receptors are distributed in a non-overlapping fashion in rat liver, and that the distribution of CI-MPR changes over a percoll gradient between newborn and adult animals. By using marker proteins it was observed that in newborns the CI-MPR is located both in intracellular fractions and in fractions that coincide with a plasma membrane marker, whereas in adults it is only detected in intracellular fractions. It was also noted that N-acetyl-ß-D-glucosaminidase distribution is closer to CI-MPR than to CD-MPR and that acid phosphatase did not match with any receptor. This evidence may also suggest that both receptors have different functions, mainly at early stages in the development of organs.

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.

Castration induces changes in the cation-dependent mannose-6-phosphate receptor in rat epididymis: possible implications in secretion of lysosomal enzymes.

It is believed that the mammalian epididymis participates in the maturation of the sperm due to its secretory activity. High concentrations of several secreted acid hydrolases are found in the epididymal lumen. Moreover, some of these enzymes are secreted by the epididymal epithelium in an androgen-dependent fashion. In this study, we attempted to discern whether mannose-6-phosphate receptors (MPRs) regulate transport and secretion of lysosomal enzymes in the rat epididymis, and if these events are altered when the animals are subjected to hormonal manipulation. We observed that expression of cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR) increased significantly in caudal epididymis of castrated rats by immunoblot. This increase was corroborated by quantitation of MPRs, by binding assays. This change could be due to androgen deprivation, as a similar effect was observed after treatment with the anti-androgenic drug flutamide. Furthermore, we observed that the CD-MPR was redistributed to the apical area of the epithelium on castrated rats by immunohistochemistry, which is compatible with the redistribution of the receptors toward lighter fractions in a Percoll gradient. Consistent with a possible involvement of the CD-MPR in the secretion, we observed an increase in pro-cathepsin D levels in epididymal fluid after castration. We conclude that the CD-MPR might be regulated by hormones and that this receptor might be involved in the secretion of specific enzymes into the rat epididymis.

Organ-specific changes in the expression of mannose-6-phosphate receptors during postnatal development in rats.

The significance of the coexistence of 2 mannose-6-phosphate receptors (MPRs) in most cell types still remains poorly understood. In this study, expression of the cation-dependent MPR (CD-MPR) and the cation-independent MPR (CI-MPR) was measured by Western blot in rat organs at 3 ages, i.e. in newborn and 10- and 90-day-old rats. It was observed that expression of the CI-MPR tends to diminish from newborns to adults in 5 of the 6 organs studied, whereas the CD-MPR did not show a clear tendency over time. In pancreas, conversely, both MPRs increased progressively from newborns to adults. The activity of 2 acid hydrolases was also measured at the different ages, and a low correlation was found with the expression of the 2 MPRs. With the exception of the pancreas, it is possible that the CI-MPR is mostly occupied with the clearance of insulin-like growth factor-II at early stages of development, and that later both MPRs may participate in the maturation of the lysosomal apparatus. We propose that in the pancreas, both receptors may be involved in increasing the proteolytic activity of this exocrine gland during postnatal development.

Targeting exogenous ß-Defensin to the endolysosomal compartment via a vehicle guided system.

A number of pathogens for which there are no effective treatments infect the cells via endocytosis. Once in the endosomes, the pathogens complete their life cycle by overriding normal lysosomal functions. Recently, our laboratory identified the lysosomal targeting signal of prosaposin, which is recognized by the sorting receptor "sortilin". Based on this evidence, we tested whether the antimicrobial peptide ß-Defensin linked to the targeting sequence of prosaposin (ßD-PSAP) could be redirected from its secretory pathway to the endolysosomal compartment. To this effect, ßD-PSAP was transfected into COS-7 cells. The sub-cellular distribution of ßD-PSAP was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that ßD-PSAP overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that ßD-PSAP was in the lysosomal fractions. In addition, our binding inhibition assay demonstrated that ßD-PSAP bound specifically to sortilin. Similarly, the delivery of ßD-PSAP was abolished after overexpressing a truncated sortilin. These results indicate that the prosaposin C-terminus and D/C-domain (prosaposin targeting sequence) was an effective "guidance system" to redirect ßD-PSAP to the endolysosomal compartment. In the future, this and other fusion proteins with antimicrobial properties will be assembled to our "biotic vehicle" to target pathogens growing within these compartments.

Changes in phosphomannosyl ligands correlate with cation-dependent mannose-6-phosphate receptors in rat liver during perinatal development.

The co-existence of two mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still a dilemma to be resolved. In this study, some parameters were measured to explore lysosomal apparatus evolution in rat liver during perinatal development, and establish a possible involvement of CD- and/or CI-MPR in lysosome maturation. Activity of four acid hydrolases was measured in the whole organ at different ages and it was found that N-acetyl-beta-D-glucosaminidase (NAG), beta-galactosidase, and beta-glucuronidase change during development, reaching a peak at the 10th day after birth. These results correlated with the expression and binding properties of CD-MPR previously reported. We also used a method that recognizes phosphomannosylated ligands by using purified biotinylated CI-MPR as a probe, and found that the highest concentrations of ligands also appear around the 10th day. Binding assays were also carried out, incubating endogenous NAG from 10-day-old and adult rats with membranes from their respective ages, and the results indicated that cation-dependent mannose-6-phosphate receptor (CD-MPR) has more impact on trafficking of the enzyme at the 10th day after birth. We concluded that lysosome maturation in the rat liver occurs around the 10th day after birth, and that the CD-MPR may participate in that event.