Limits...
The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.

Barisone GA, Albertali IE, Sánchez M, Cabada MO - Reprod. Biol. Endocrinol. (2003)

Bottom Line: Galactose residues are present mainly in gp120 of FE.Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface.The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Area Biología-Facultad de Ciencias Bioquímicas y Farmaceuticas-UNR and Cellular Biology (CONICET-UNR), Suipacha 531, Rosario S2002LRK, Argentina. gbarison@fbioyf.unr.edu.ar

ABSTRACT
A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

Show MeSH

Related in: MedlinePlus

Lectin blot analysis of CE , VE , and FE . Solubilized envelopes were resolved by SDS-PAGE under reducing conditions (10% gels, 5 μg protein/lane) and electrotransferred to nitrocellulose. Membranes were incubated with peroxidase-conjugated lectins and developed as indicated in Materials and Methods. Controls (only shown for HAA) were carried out by incubating the lectin with the corresponding haptenic sugar prior to membrane incubation. Molecular mass markers are indicated on the left. Images are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC153491&req=5

Figure 3: Lectin blot analysis of CE , VE , and FE . Solubilized envelopes were resolved by SDS-PAGE under reducing conditions (10% gels, 5 μg protein/lane) and electrotransferred to nitrocellulose. Membranes were incubated with peroxidase-conjugated lectins and developed as indicated in Materials and Methods. Controls (only shown for HAA) were carried out by incubating the lectin with the corresponding haptenic sugar prior to membrane incubation. Molecular mass markers are indicated on the left. Images are representative of three independent experiments.

Mentions: The results summarized in Figure 3 indicate that Con A binds to gp120 in the three envelopes: CE , VE and FE (Fig. 3A). This indicates that this component likely contains mannose or glucose residues. PNA staining revealed the presence of galactose (or N-acetyl-galactosamine) residues in FE gp120. Much weaker staining is also observed for gp120 in CE and VE (Fig. 3B). No other components seem to bind the lectin, indicating the absence of terminal galactose residues. Treatment with WGA revealed the presence of sialic acid and probably N-acetyl-glucosamine in all the components of the three envelopes, although a higher amount seems to be associated with gp120 (Fig. 3C). N-acetyl-galactosamine is present in all the components of the three envelopes (as evidenced by HAA binding), except for gp69 in FE , which was not stained with this lectin (Fig 3D). This could be ascribed to the loss of N-acetyl-galactosamine residues due to the activity of enzymes released from the CGs during activation. It should be noted that in this case pre-treatment of the lectin with D-glucosamine only partially inhibits the binding. This is frequently observed when binding inhibition is carried out with a monosaccharide instead of the more complex (and specific) oligosaccharide. In any case, the staining in control experiments is weaker (Fig. 3D), indicating that, at least to some extent, the lectin specifically binds to N-acetyl-galactosamine residues in the envelope glycoproteins.


The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.

Barisone GA, Albertali IE, Sánchez M, Cabada MO - Reprod. Biol. Endocrinol. (2003)

Lectin blot analysis of CE , VE , and FE . Solubilized envelopes were resolved by SDS-PAGE under reducing conditions (10% gels, 5 μg protein/lane) and electrotransferred to nitrocellulose. Membranes were incubated with peroxidase-conjugated lectins and developed as indicated in Materials and Methods. Controls (only shown for HAA) were carried out by incubating the lectin with the corresponding haptenic sugar prior to membrane incubation. Molecular mass markers are indicated on the left. Images are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153491&req=5

Figure 3: Lectin blot analysis of CE , VE , and FE . Solubilized envelopes were resolved by SDS-PAGE under reducing conditions (10% gels, 5 μg protein/lane) and electrotransferred to nitrocellulose. Membranes were incubated with peroxidase-conjugated lectins and developed as indicated in Materials and Methods. Controls (only shown for HAA) were carried out by incubating the lectin with the corresponding haptenic sugar prior to membrane incubation. Molecular mass markers are indicated on the left. Images are representative of three independent experiments.
Mentions: The results summarized in Figure 3 indicate that Con A binds to gp120 in the three envelopes: CE , VE and FE (Fig. 3A). This indicates that this component likely contains mannose or glucose residues. PNA staining revealed the presence of galactose (or N-acetyl-galactosamine) residues in FE gp120. Much weaker staining is also observed for gp120 in CE and VE (Fig. 3B). No other components seem to bind the lectin, indicating the absence of terminal galactose residues. Treatment with WGA revealed the presence of sialic acid and probably N-acetyl-glucosamine in all the components of the three envelopes, although a higher amount seems to be associated with gp120 (Fig. 3C). N-acetyl-galactosamine is present in all the components of the three envelopes (as evidenced by HAA binding), except for gp69 in FE , which was not stained with this lectin (Fig 3D). This could be ascribed to the loss of N-acetyl-galactosamine residues due to the activity of enzymes released from the CGs during activation. It should be noted that in this case pre-treatment of the lectin with D-glucosamine only partially inhibits the binding. This is frequently observed when binding inhibition is carried out with a monosaccharide instead of the more complex (and specific) oligosaccharide. In any case, the staining in control experiments is weaker (Fig. 3D), indicating that, at least to some extent, the lectin specifically binds to N-acetyl-galactosamine residues in the envelope glycoproteins.

Bottom Line: Galactose residues are present mainly in gp120 of FE.Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface.The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Area Biología-Facultad de Ciencias Bioquímicas y Farmaceuticas-UNR and Cellular Biology (CONICET-UNR), Suipacha 531, Rosario S2002LRK, Argentina. gbarison@fbioyf.unr.edu.ar

ABSTRACT
A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

Show MeSH
Related in: MedlinePlus