ABSTRACT
Guanidinobenzoatase (GB), a serine proteinase with a molecular weight of 71,000, is found both free in the epididymal fluids of the mouse and bound to the sperm surface. Microgram quantities of the enzyme, purified from epididymal fluid, will completely disperse follicle cells from freshly ovulated oocytes after 15 min of incubation. Purified GB exhibits no hyaluronidase activity as determined by the acid albumin assay. The ability of GB to disperse follicle cells is blocked by a proteinase inhibitor endogenous to the male reproductive tract. The inhibitor has no effect on bovine testicular hyaluronidase. Although the function of GB has not been defined, the observations presented here indicate that it may play a role in cumulus matrix penetration during fertilization.
Subject(s)
Carboxylic Ester Hydrolases/physiology , Endopeptidases/physiology , Epididymis/enzymology , Oocytes/metabolism , Oogenesis , Animals , Carboxylic Ester Hydrolases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Endopeptidases/isolation & purification , Epididymis/chemistry , Extracellular Matrix/metabolism , Female , Hyaluronoglucosaminidase/metabolism , Male , Mice , Oocytes/cytologyABSTRACT
Guanidinobenzoatase (GB), a proteolytic enzyme found in the epididymal fluids of mice, was purified to apparent homogeneity by molecular sieving and affinity chromatography. It has a molecular mass of 71 kDa and its enzymatic activity is heat labile and sensitive to EGTA. Its kinetic parameters (K(m) of 6.66 microM and a Vmax of 4.38 nmol/min/mg) were determined using 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) as the substrate. GB activity is concentrated in the cauda epididymal region of the genital tract. Heat-solubilized whole zonae, biologically active ZP3, and several serine proteinase inhibitors, including a proteinase inhibitor endogenous to the male genital tract, effectively block the ability of GB to hydrolyze MUGB. Pretreating cumulus-free, zonae intact oocytes with purified GB reduces, in a concentration-dependent manner, the number of sperm able to bind to the zonae. The function of the soluble enzyme is not known. Its ability to bind both trypsin inhibitors and ZP3 suggests a possible role in gamete recognition.
Subject(s)
Carboxylic Ester Hydrolases/isolation & purification , Endopeptidases/isolation & purification , Epididymis/chemistry , Receptors, Cell Surface , Acrosome/physiology , Animals , Body Fluids/chemistry , Carboxylic Ester Hydrolases/metabolism , Chelating Agents/pharmacology , Chromatography, Affinity , Chromatography, Gel , Egg Proteins/metabolism , Egg Proteins/pharmacology , Egtazic Acid/pharmacology , Endopeptidases/metabolism , Female , Kinetics , Male , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred ICR , Serine Proteinase Inhibitors/pharmacology , Sperm-Ovum Interactions , Trypsin Inhibitors/pharmacology , Zona Pellucida/metabolism , Zona Pellucida GlycoproteinsABSTRACT
Mouse caput spermatozoa are considered immature and thus unable to fertilize oocytes. In this study, we determined whether washing mouse caput spermatozoa increased their ability to acrosome react in response to a physiological stimulus. The results obtained showed that mouse caput spermatozoa incubated in Earles' modified medium containing calcium chloride and supplemented with BSA and pyruvate for 1 h at 37 degrees C and then washed acrosome reacted in response to both solubilized zonae and immunoaggregation of a zona binding site. In addition, the material removed from caput spermatozoa by washing blocked induced acrosome reactions of cauda spermatozoa. The data indicate that mouse caput epididymal spermatozoa, if incubated and washed, can undergo physiological acrosome reactions.
Subject(s)
Acrosome/physiology , Epididymis/physiology , Fertilization/physiology , Animals , Cells, Cultured , Exocytosis/physiology , Female , Male , Mice , Mice, Inbred ICR , Sperm Capacitation/physiology , Zona Pellucida/physiologyABSTRACT
Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight approximately 20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization.
