ABSTRACT
BACKGROUND: Post-diluents could potentially increase semen cryotolerance, but remain poorly explored in horses. OBJECTIVE: The aim was to evaluate the efficiency of post-diluents on frozen-thawed semen viability of two stallions (S1-S2). MATERIALS AND METHODS: The cryopreserved semen was thawed at 50°C for 40 seconds. Semen motility and acrosin activity (AA) were determined during the thermo-resistance test (TRT). RESULTS: Progressive motility of S2 semen decreased after 60 and 90 minutes of TRT (TRT60 and TRT90) on the control compared to both post-diluents. The total motility of both S1 and S2 decreased on TRT60 and TRT90 semen control versus both Ringer and Merk post-diluents. The AA on S1 was higher than S2 throughout the TRT. Pregnancy rates after artificial insemination (AI) were similar among post-diluents and stallions. CONCLUSION: Post-diluents do not contribute to predicting frozen-thawed semen fertility or the efficiency of equine AI.
Subject(s)
Acrosin/chemistry , Cryopreservation/veterinary , Horses , Semen Preservation/veterinary , Sperm Motility , Animals , Female , Fertility , Freezing , Insemination, Artificial , Male , Pregnancy , Semen , SpermatozoaABSTRACT
OBJECTIVE: To characterize proacrosin/acrosin interaction with isolated zona pellucida (ZP) components. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10, and Rec-6) and from human ZP glycoproteins (rec-hZPA, ZPB, and ZPC). INTERVENTION(S): In vitro binding assay developed to assess proacrosin/acrosin-ZP interaction. MAIN OUTCOME MEASURE(S): Zona pellucida glycoprotein binding to proacrosin/acrosin; estimation of binding affinity. RESULT(S): Of all ZP proteins, rec-hZPA demonstrated the highest binding activity toward acrosin (Rec-30) (rec-hZPB: 42% of rec-hZPA; rec-hZPC: 39% of rec-hZPA; P<.0005). Rec-hZPA interaction was disturbed by dextran sulphate (75% inhibition with 10 microM), fucose (67% inhibition with 1.5 microM), and mannose (69% inhibition with 333 mM). Comparing binding activity of proacrosin with other N-terminal acrosin fragments, Rec-40 showed 2.6-3 times higher levels. Moreover, saturable high affinity binding of Rec-40 to ZP components was observed (Kd: 34 nM for rec-hZPA, 38 nM for rec-hZPB, 63 nM for rec-hZPC). CONCLUSION(S): The rec-hZPA is the major ZP ligand for human proacrosin/acrosin. The interaction involves mannosyl, fucosyl, and sulfated glycans. Binding sites for rec-hZP would be located both at the N- and C-terminus of proacrosin, revealing a key role of the proenzyme in the interaction.
Subject(s)
Acrosin/metabolism , Egg Proteins/metabolism , Enzyme Precursors/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Zona Pellucida/enzymology , Acrosin/chemistry , Acrosin/physiology , Animals , Binding Sites/physiology , CHO Cells , Cricetinae , Enzyme Precursors/chemistry , Enzyme Precursors/physiology , Humans , Male , Prospective Studies , Protein Binding/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Zona Pellucida/metabolism , Zona Pellucida/physiology , Zona Pellucida GlycoproteinsABSTRACT
OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.
Subject(s)
Acrosin/immunology , Antibodies/pharmacology , Enzyme Precursors/immunology , Fertilization in Vitro , Peptide Fragments/immunology , Sulfates/metabolism , Acrosin/chemistry , Acrosin/physiology , Amino Acid Sequence , Animals , Antibodies/metabolism , Binding Sites/immunology , Enzyme Precursors/chemistry , Enzyme Precursors/physiology , Female , Fertilization/physiology , Fluorescent Antibody Technique , Male , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Polysaccharides/metabolism , Sperm Capacitation , Zona Pellucida/physiologyABSTRACT
Acrosin is an acrosomal protease synthesized as a proenzyme and activated into beta-acrosin during the acrosome reaction. In the present study, a set of sensitive assays was developed to identify the proacrosin/acrosin system and to evaluate its activation pattern in human sperm extracts. Immunocytochemical analysis with monoclonal antibody (Mab) AcrC5F10 showed specific staining on the acrosome of permeabilized ejaculated and capacitated spermatozoa. Acrosome reaction was associated with a decrease in staining. AcrC5F10 specifically recognized a 55-kDa band (proacrosin) in Western immunoblots. Activation studies showed enzymatically active intermediates of 39 and 35 kDa after zymography. Immunoreactive bands of 52, 43, 34, 21-26 and 16 kDa were identified in the activation patterns developed with AcrC5F10. Activation was completely inhibited in the presence of 9 mM CaCl(2) or 100 mM benzamidine. A multiple sequence alignment revealed partial conservation of putative cleavage sites in the proacrosin sequence. The tests described allow the detection of human proacrosin in spermatozoa and sperm protein extracts, as well as the evaluation of the proenzyme activation pattern. They can be used to study the effect of inhibitors upon proenzyme activation. In addition, alterations in proacrosin activation in semen samples with abnormal acrosin enzymatic activity can be analyzed using these assays.
Subject(s)
Acrosin/metabolism , Enzyme Precursors/metabolism , Spermatozoa/enzymology , Acrosin/chemistry , Amino Acid Sequence , Animals , Enzyme Activation , Enzyme Precursors/chemistry , Humans , Hydrogen-Ion Concentration , Immunohistochemistry , Male , Molecular Sequence Data , Molecular WeightABSTRACT
Proacrosin is the zymogen of acrosin, a serine protease localized in the acrosomal matrix of mammalian sperm. Proacrosin/acrosin binds to solubilized zona pellucida glycoproteins (ZPGs) and various polysulfates in a non-enzymatic mechanism. In addition, both polysulfates and ZPGs induce proacrosin activation once they bind to the polysulfate-binding domain (PSBD) of the enzyme. We show here that the peptide (43)IFMYHNNRRYHTCGGILL(60) inhibited the proacrosin activation induced by either fucoidan or ZPGs. In addition, the peptide was recognized by the monoclonal antibody C5F10, which is directed against the PSBD region. Our data suggest that the PSBD is composed of many "subsites" that may or may not interact with each other.