Epididymal α- l -fucosidase and its possible role in remodelling the surface of bull spermatozoa (original) (raw)
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Biochemical Journal, 1998
In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum α--fucosidase in rat sperm. In the present study, rat sperm α-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of α--fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53p2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of α-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. α--Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and α--fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5n2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the
The Biochemical journal, 1998
In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum alpha-L-fucosidase in rat sperm. In the present study, rat sperm alpha-L-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of alpha-L-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53+/-2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of alpha-L-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. alpha-L-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and alpha-L-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sper...
Biology of Reproduction, 2001
Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le a trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le a trisaccharide (␣-L-Fuc[1,4]--D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le a-binding protein with an apparent mass of 16.5 kDa. Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.
Evidence for a fucose-binding protein in boar spermatozoa
Histochemistry, 1985
A fucose binding protein was detected in boar spermatozoa by means of a specifically developed modified enzyme-linked-lectin-assay using glycosylated peroxidase derivatives. The distribution of the fucose binding protein was assessed by means of fluorescence microscopy with fluoresceinyl-glycosylated peroxidase. Fucose binding was particularly prominent at the apical region of the sperm head. In order to gain more insight into the precise localization of the carbohydrate binding protein electron microscopical studies were performed using fucosyl peroxidase coupled to colloidal gold. In ultrathin sections as well as in specimens prepared in toto for TEM an intensive binding of fucosylperoxidase-colloidal gold was predominantly found at the apical part of the acrosome appearing as a crescent-like area. In some cases this binding pattern was replaced by a triangle-like intensive labelling at the equatorial segment as revealed clearly by specimens prepared in toto. By SDS-PAGE of the SDS-extractable sperm-proteins, followed by transblotting to nitrocellulose and visualization with the fucosylperoxidase by enzymatic amplification with 4-chloro-l-naphthol mainly one protein with the reduced molecular weight of approximately 53 kdal and some small proteins with apparent molecular weights less than 20 kdal was found to be responsible for the fucosebinding ability of porcine spermatozoa.
Physiological State of Bull Sperm Affects Fucose- and Mannose-Binding Properties
Biology of Reproduction, 2000
In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to ␣-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean ؎ SD:167 ؎ 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 ؎ 2.7) and in washed sperm with seminal plasma added back (56 ؎ 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 ؎ 6.3) and by capacitating washed sperm in medium containing 10 g/ml heparin (50 ؎ 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 ؎ 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 ؎ 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.
Biochemical and Biophysical Research Communications, 1997
subsequent steps leading to successful fertilization. The male reproductive system of the mollusc bivalve There is also evidence that other glycosidases such as Unio elongatulus contains two distinct forms of a-Lb-N-acetylglucosaminidase (6-9) and a-D-mannosidase fucosidase, one present in the gonad fluid and a second (10) play a similar role in fertilization. In all cases, the one associated with the sperm plasma membrane. Both glycosidase on the sperm has been found to match the activities were purified to homogeneity. The soluble functional sugar residues of the egg coat glycoproteins. seminal plasma enzyme had an oligomeric MW of 56 In a previous study, we showed that the ligand for kDa as determined by MALDI-TOF mass spectrometry, sperm binding in the mollusc bivalve Unio elongatulus whereas the enzyme purified from sperm plasma memis a 220 kDa glycoprotein and that fucose residues of its branes had an MW of 68 kDa. Analyzed by lectin blotoligosaccharide chains play a key role in the interaction ting with ConA and PNA, the 68 kDa enzyme did not mechanism (11,12). We postulated that a-L-fucosidase bind either lectin, whereas the 56 kDa form bound could be a receptor molecule on the sperm. In this re-ConA only. Both fucosidases followed a Michaelis-Menport, we demonstrate that two forms of a-L-fucosidase, ten kinetics with the K m of the sperm-bound enzyme one soluble and the other bound to the sperm membeing 7.1 1 10 04 M and that of the seminal enzyme being brane, are present in the gonads of Unio elongatulus. 9.1 1 10 04 M. Both had a pH optimum of 5.0. ᭧ 1997 Academic Press
Biology of Reproduction, 1987
Rat spermatozoa were recovered from the cap Ut, Corp US, and cauda epididymides and assayed for glycosidase activity, total nonamino (neutral) carbohydrate, and protein content. The activities of 13-glucosidase, #{216}-galactosidase, 13-N-acetylglucosaminidase, and 13-N-acetylgalactosaminidase were fluorometrically assayed in spermatozoa and membrane-enriched fractions. Except for j3-glucosidase, the activities of the glycosidases based on protein content were greatest in whole sperm and membrane-enriched fractions obtained from the cauda epididymides. Based on sperm concentration, however, glycosidase activities increased proceeding from the caput to the corpus epididymides, then declined from the corpus to the cauda epididymides. Analyses of nonamino carbohydrate and protein content based on sperm number indicated regional trends similar to those of glycosidase activity. Total nonamino carbohydrate and protein content were highest in corpus sperm, and lowest in cauda sperm. These data indicate major quantitative changes in cell surface carbohydrate as spermatozoa traverse the epididymis. A positive correlation for the membrane-enriched fraction between increasing glycosidase activity and decreasing carbohydrate and protein content suggests that glycosidases may play a significant role in modifying the spermatozoon surface during epididymal transit and maturation.
Fucosylation Events during Mammalian Spermatogenesis b
Annals of the New York Academy of Sciences, 1987
Surface glycoproteins are involved in many aspects of cellular differentiation and interaction. Biological systems employing membrane glycoconjugates as determinants of cellular recognition processes include myogenesis,' retinal development,2 and hematop~iesis.~ Interactive events at the level of individual cells also employ surface glycocongugate mediators, as exemplified by slime mold associations: cancer metastasis,' and cellular pinocytosis.6 Because spermatogenesis in mammals entails highly orchestrated cellular interactions, similar in many respects to those cited above, it is probable that specific alterations in plasma membrane glycoproteins and glycolipids play important regulatory functions during the differentiation of the spermatozoon. Current evidence, in fact, demonstrates that a variety of changes in germ-cell surface carbohydrate moieties do occur. Furthermore, in many instances these alterations occur sequentially and exhibit restricted temporal distributions during spermatogenesis. Cells early in meiotic prophase express surface antigenic determinants recognized by monoclonal antibodies specific for high molecular weight glycoc~njugates.~ In addition, these particular antigenic determinants are progressively masked by the addition of sialic acid residues on germ-cell surfaces as Funding for these experiments was provided by Grants to C.F. Millette (HD-11267, HD-15269) and to D.R. Armant (HD-21326).
Biology of Reproduction, 1993
In previous studies, we reported that rat epididymal fluid acid D-D-galactosidase, which optimally cleaves a synthetic substrate (PNP [3-D-galactoside) at pH 3.5, shows maximum activity at pH 6.8 when a glycoprotein is used as a substrate [Skudlarek MD, Tulsiani DRP, Orgebin-Crist M-C. Biochem J 1992; 286: 907-914]. We now describe a similar pH-dependent substrate preference for rat sperm-D-galactosidase. We found that only 10-14% of total-D-galactosidase (and other glycosidase) activity was associated with spermatozoa. The remaining enzyme activities were present in soluble form in the luminal fluid. When the glycosidase levels were expressed per 106 sperm, all enzymes showed a progressive increase in spermatozoa from the caput to the corpus or proximal cauda followed by a sharp decline in spermatozoa from the distal cauda epididymidis. The observed decrease in P-D-galactosidase activity could not be explained by the loss of cytoplasmic droplets (which have a low enzyme activity relative to spermatozoa) or the presence of inhibitors/activators of the enzyme activity in spermatozoa from the proximal or distal epididymis. However, we found that the changes in-D-galactosidase activity during sperm maturation in the epididymis were accompanied by changes in the molecular form(s) of the enzyme. Western blot analysis using an antibody to D1-D-galactosidase showed a progressive processing of the 82-kDa immunoreactive band in caput spermatozoa to an 80-kDa diffuse band in cauda spermatozoa. The sperm-associated-D-galactosidase form(s) does not appear to be due to adsorption and/or binding of the luminal fluid-D-galactosidase, which contained a 97-kDa form in fluid from the caput and two forms, of 97 kDa and 84 kDa, in corpus and cauda fluids. The observed difference in the molecular forms of the sperm and luminal fluid was found to be due to differential glycosylation, since deN -glycosylation of various forms of P-D-galactosidase generated a single immunoreactive form of 70 kDa. Subcellular localization studies and assay for the-D-galactosidase activity in the enriched plasma membrane and acrosomal membrane fractions suggested the likelihood that the activity of [{-D-galactosidase and other glycosidases is present in the acrosome and is readily released during sperm disruption. The evidence suggests that sperm-D-galactosidase may be functional within the acidic environment of the acrosome during sperm maturation as well as in the neutral environment of the oviduct after the zona-induced acrosome reaction.
Theriogenology, 2017
Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-L-fucosidase, α-D-galactosidase, β-D-galactosidase, β-D-Nacetylhexosaminidase, and α-D-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-D-mannosidase, which was increased (P<0.001), and β-Dgalactosidase, which was decreased (P<0.001) at neutral pH, and of α-D-galactosidase and β-Nacetylhexosaminidase, which were decreased (P<0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both β-D-galactosidase and to a lesser degree α-D-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.