Binding of Lectins to the Zona Pellucida of In Vitro Matured Pig Oocytes and Sperm-Oocyte Interaction In Vitro.

Abstract

Immature pig oocytes cultured for 36 h in a modified tissue culture medium 199B were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescence illumination, Ricinus communis agglutinin (RCA-I) and Lens culinaris agglutinin (LCA) bound to all oocytes, with the strongest fluorescence in either the outer region of the ZP (RCA-I) or throughout the ZP (LCA). However, Bandeiraea simplicifolia lectin-I (BS-I) and Ulex europaeus agglutinin-I (UEA-I) bound with either strong or weak intensity mainly to the outer and inner regions of the ZP, respectively, in ~70-90% of oocytes examined. Bandeiraea simplicifolia lectin-II (BS-II) bound to various regions of the ZP with different intensities in only 60% of oocytes examined. At 2 h after insemination in vitro, significantly fewer spermatozoa were bound to the ZP of lectin-treated oocytes than untrearted control oocytes. Of the lectins, RCA-I and LCA most inhibited sperm binding. At 12 h after insemination, penetration rates were similar in control oocytes and those treated with BS-I or BS-II, but penetration rates were lower in oocytes treated with UEA-I than those in untreated controls, and an almost complete block of sperm penetration was observed in oocytes treated with RCA-I or LCA. The incidence of monospermy was similar in untreated oocytes and those treated with BS-I or UEA-I, but it was higher in oocytes treated with BS-II. These results suggest that β-D-galactose and α-D-mannose residues in the pig ZP may act as primary sperm receptors and/or inducers of the sperm acrosome reaction and β-D-N-acetylglucosamine residues may be involved in the block of polyspermy. The variability in binding of BS-II to the ZP, which correlated with monospermy/polyspermy may reflect differences in the maturation state of individual oocytes. In future, this lectin might provide a means of monitoring maturation in vitro, leading to the development of improved culture systems. Key words: Pig, Oocyte, Zona pellucida, Lectin-binding, Gamete interaction.The zona pellucida (ZP), a relatively thick extracellular coat that surrounds mammalian oocytes, is known to mediate critical steps in fertilization including speciesspecific binding of spermatozoa, inducing the sperm acrosome reaction and preventing penetration of excess spermatozoa (reviewed in [1,2]). The mammalian ZP typically consists of a few glycoproteins which have different polypeptide chains and oligosaccharides [3][4][5][6][7][8] and evidence indicates that these oligosaccharides play an important role in sperm-oocyte recognition in all mammalian species (reviewed in [9][10][11]). Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity [12]. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the ZP of several mammalian species [13][14][15][16][17] including...

Identification, Localization and Involvement of Glycosidases in Sperm-Zona Interaction Using Frozen-Thawed Ejaculated Pig Spermatozoa.

“…Although it has been reported that pig spermatozoa which initially bind to the ZP have intact plasma membrane [30][31][32], the present results indicate that, in the pig, only spermatozoa undergoing or completing acrosome reaction could bind firmly to the ZP as suggested by Jones et al [33]. However, it is unclear at present how α-L-fucosidase might be involved in binding of spermatozoa to the ZP because it has been shown that fucose residues are present only in the inner region of the ZP [23]. It has been reported in mice that α-galactosyl residues which are present in the inner region of the ZP [34] also play a role as a sperm receptor [35].…”

“…It is known that the penetration of spermatozoa through the ZP may be facilitated by hydrolytic enzymes released from the acrosome (for review see [36]). Furthermore, it has been shown that abundant Man and Gal residues are present throughout the ZP and in the outer region of the ZP, respectively, while Fuc residues are found only in the inner region of the ZP in pig oocytes [23]. Therefore, it is possible that α-L-fucosidase, α-Dmannosidase and β-D-galactosidase are all involved in zona penetration of spermatozoa and that each glycosidase plays a role in the different regions of the ZP.…”

“…It has been reported that a 55 kD protein found in the plasma membrane [19] and a 53 kD protein in the acrosome [20][21][22] in pig spermatozoa are implicated in sperm-zona interaction. We recently found that α-L-fucose (Fuc), β-D-galactose (Gal), α-D-mannose (Man) and N-acetyl-β-D-glucosamine (GlcNAc) residues are all present with different amounts and distributions in the ZP of pig oocytes, and Gal and Man residues appear to play important roles in initial sperm binding and subsequent penetration through the ZP [23]. We have hypothesized that glycosidases specific to these sugar residues in the ZP are present in pig spermatozoa and may play roles in sperm-zona interaction.…”

α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa

“…Fucose has been detected in the porcine ZP using Ulex europaeus agglutinin 1 (UEA-I) lectin histochemistry (Song et al, 1999;Pastor et al, Pattern A showed significant differences, with higher values observed in the control group (54.7 ± 1.6%) than in the TALP (15.1 ± 1.2%) and TALP + E (12.2 ± 1.1%) groups (P < 0.001); pattern B showed higher values in the TALP group (61.8 ± 1.6%) than in the TALP + E (35.2 ± 1.6%) and control (43.2 ± 1.6%) groups (P < 0.01); pattern C showed significant differences (P < 0.001), with maximum values observed in the TALP + E group (52.5 ± 1.7%) compared with the TALP (23.1 ± 1.4%) and control (2.1 ± 0.5%) groups. Different letters indicate significant differences.…”

“…It shows maximum activity in pig OF close to the moment of ovulation (Carrasco et al, 2008b). Its substrate, fucose, has been detected in sugar chains on the spermatozoal surface in the mouse (Boldt et al, 1989), bull (Taitzoglou et al, 2007) and boar (Jiménez et al, 2003), and also on the inner region of the ZP in pigs (Song et al, 1999). In addition, when carbohydrate involvement in spermatozoaegg interactions were studied by adding sugars to fertilisation media, fucose and the polysaccharide fucoidan induced a significant drop in penetration rates in cattle (Tanghe et al, 2004) and pigs (Töpfer-Petersen et al, 1985;Song et al, 2007b).…”