Effect of N-Acetyl-D-Glucosamine on Bovine Sperm-Oocyte Interactions

Abstract

Abstract. N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in spermoocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to coincubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/ propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization. Key words: Bovine, Fertilization, Monosaccharides, Sperm, Zona pellucida (J. Reprod. Dev. 55: [676][677][678][679][680][681][682][683][684] 2009) ertilization of oocytes comprises several interactions between the sperm and oocyte. Contact between the sperm and oocyte first occurs at the outer surface of the surrounding extracellular matrix of the egg, the zona pellucida (ZP). Binding of the lectinlike protein on the sperm head to distinct oligosaccharides of ZP glycoproteins leads the capacitated sperm to recognize the oocyte. Upon anchoring of the sperm to the oocyte through the ZP carbohydrates, a signaling cascade induces the acrosome reaction of the sperm, allowing the sperm to pass through the ZP. The acrosomereacted, penetrated sperm fuses with the egg plasma membrane, which enables the egg to complete meiosis, and initiates mechanisms to prevent polyspermic fertilization by the exocytosis of cortical granules (CG) [1]. Oligosaccharides on the ZP include some monosaccharides, such as N-acetyl-D-glucosamine (GlcNAc), D-mannose and D-fucose [2], and these monosaccharides are involved in many sperm-oocyte in...

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

“…Inhibition assays : between all molecules tested, β‐ N ‐acetylglucosamine blocked the binding of both β‐ N ‐acetylglucosaminidases to the eggshell (Figure F and I). A similar effect of inhibition with GlcNAc was observed in vitro in several organisms, such as hamster (Zitta et al., ), bovine (Sakaguchi et al., ), chicken (Robertson, Wishart, & Horrocks, ), and humans (Miranda et al., ). In addition, when the enzymes were preincubated with ovalbumin and transferrin at 1 mg/ml the binding was completely inhibited.…”

“…In the second time, a portion of purified β‐ N ‐acetylglucosaminidases were biotinylated using Sulfo‐NHS biotin, a simple system that does not change enzyme activity and allows one to monitor interactions between protein and carbohydrate moieties on a nitrocellulose membrane. We have carried out assays with glycoproteins carrying N ‐linked glycans with or without terminal β‐ N ‐acetylglucosamine residue, since it has been proposed the participation of β‐ N ‐acetylglucosamine in sperm–oocyte interactions in various mammalian species, mouse (Wu & Sampson, ), hamster (Zitta et al., ), bovine (Sakaguchi et al., ), and human included (Clark, , ; Miranda et al., ). We tested different carbohydrate moieties, and numerous molecules containing or not containing β‐ N ‐acetylglucosamine residues (Table ).…”

“…In particular, in insects N ‐acetylglucosamine is the monomer unit of the chitin polymer. Several works have demonstrated that N ‐acetylglucosamine residues are present on the gamete surface and might play an important role in sperm–egg recognition (Clark, , ; Sakaguchi, Iwata, Kuwayama, & Monji, ; Wu & Sampson, ; Zitta, Wertheimer, & Miranda, ). Sperm‐associated β‐ N ‐acetylhexosaminidases, which have been suggested to be involved in fertilization, have been found in many species beyond Drosophila , such as ascidians (Downey & Lambert, ; Godknecht & Honegger, ; Hoshi, ; Hoshi et al., ; ), amphibians (Martinez, Martelotto, & Cabada, ; Prody, Greve, & Hedrick, ), and mammals, humans included (Miranda et al., ; Perez Martinez et al., ; Zitta et al., ).…”

Metabolite Support of Long-Term Storage of Sperm in the Spermatheca of Honeybee (Apis mellifera) Queens

“…After transfer into the spermatheca, stored sperm is generally assumed to be inactive until activated during fertilization by Zrt-and Irt-like protein transporter 7.1 (ZIPT-7.1) (Zhao et al, 2018) (not detected in the present study) and mineral zinc (Chu, 2018). Sakaguchi et al (2009) studied bovine sperm-oocyte interactions and discovered that supplementary N-acetyl-Dglucosamine (GlcNAc) (at 5 or 25 mM) significantly suppresses the number of sperm that attach to the zona pellucida (Sakaguchi et al, 2009). Moreover, a recommended concentration of 0.05% soluble GlcNAc significantly increases the cryopreservation of boar spermatozoa (Yi et al, 2002).…”

“… Sakaguchi et al (2009) studied bovine sperm–oocyte interactions and discovered that supplementary N-acetyl-D-glucosamine (GlcNAc) (at 5 or 25 mM) significantly suppresses the number of sperm that attach to the zona pellucida ( Sakaguchi et al, 2009 ). Moreover, a recommended concentration of 0.05% soluble GlcNAc significantly increases the cryopreservation of boar spermatozoa ( Yi et al, 2002 ).…”