title: Posttranslational processing of PH-20 during epididymal sperm maturation in the horse.
abstract: It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.
authors.affiliation: Department of Anatomy and Embryology, School of Veterinary Medicine, Autonomous University of Barcelona, Barcelona 08193, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Rutllant
authors.forename: S A
authors.initials: SA
authors.lastname: Meyers
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006736:Horses; D006821:Hyaluronoglucosaminidase / Q000032:analysis; D008297:Male; D008856:Microscopy, Fluorescence; D011499:Protein Processing, Post-Translational; D013091:Spermatogenesis; D013094:Spermatozoa / Q000737:chemistry / Q000201:enzymology / Q000502:physiology*
chemical_list: D015815:Cell Adhesion Molecules; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20