The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

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.

Figure 1

Electrophoretic profile of the coelomic envelope (CE) as compared with the vitelline envelope (VE). Solubilized glycoproteins were separated on a 10% gel under denaturing and reducing (+) or non-reducing (-) conditions. Forty μg of protein was loaded in each lane. The gel was stained with Coomasie Brilliant Blue R-250 (CBB, lanes 1–3) for proteins or with the periodic acid-Schiff protocol (PAS, lane 4) for carbohydrates. Molecular mass markers are indicated on the left. Arrowheads point to distinct CE components, which are not found in the VE . βME: β-mercaptoethanol. Images are representative of four independent experiments.

Figure 2

A: Electrophoretic analysis of deglycosylated components of the vitelline (VE) and fertilization envelopes (FE). Solubilized envelopes before (VE , FE) or after elimination of N- and O-linked oligosaccharides (dVE, dFE) were analyzed on a 12% gel under denaturing and reducing conditions. Forty μg of protein was loaded in each lane. The gel was stained with Coomasie Brilliant Blue R-250. Arrowheads point to the components that differ between the two envelopes. B: Electrophoretic analysis of the FE under denaturing and reducing (+) or non-reducing (-) conditions. Forty μg of protein was loaded in each lane (10% gel), and was then stained for proteins (CBB) or carbohydrates (PAS). Numbers indicate molecular mass markers. βME: β-mercaptoethanol. Images are representative of four independent experiments.

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.

Figure 4

Lectin binding to whole oocytes and embryos. FITC-labeled lectins were used to analyze carbohydrates on the surface of VE and FE . Ten to twenty eggs were incubated with the lectin, washed, and observed under a fluorescence-inverted microscope. Controls were carried out as for lectin blots. Results are shown for ConA (a-d), PNA (e-h), HAA (i-l) and WGA (m-p). Micrograph exposure times were the same in all cases, except for j, which was over-exposed to record the observations described. "Vis" (q-t) indicates eggs photographed under visible light. Images are representative of four independent experiments.

Figure 5

Electrophoretic analysis of VE (lane 1), VE post incubation with CGP (lane 2) and FE (lane 3). Total proteins, 20 μg, of VE , treated VE and FE were run in SDS-PAGE 7.5%, under reducing conditions and silver stained. VE is hydrolyzed by CGP , only the gp75 changes its electrophoretic mobility from 75 kDa to 69 kDa. Molecular mass markers are displayed on the left of the gel (numbers indicate the molecular mass in kDa). Images are representative of five independent experiments.

Figure 6

A, Hydrolytic activity of CGP on VE under different pH's. VEs were incubated with 5 μg of CGP (total protein) in the same conditions but different pH's. Lane 1, pH 4.0; Lane 2, pH 5.6; Lane 3, pH 7.0; Lane 4, pH 7.6; Lane 5 pH 8.1; Lane 6, pH 9.0; Lane 7, pH 10.0, Lane 8, VE; Lane 9, FE . The products were analyzed by SDS-PAGE 7.5 %. CGP shows hydrolytic activity at a pH between 5.6 and 8.1. The gel was stained with Coomassie blue. B: Hydrolytic activity of CGP on VE under different protease inhibitors. VEs were incubated with 5 μg total proteins of CGP . Lane 1, calcium free; Lane 2, 5 mM EDTA; Lane 3, 0.7 μg/ml pepstatin; Lane 4, 50 μg/ml TLCK; Lane 5, 50 μg/ml TPCK; Lane 6, VE ; Lane 7, FE ; Lane 8, AE (obtained by treating the VE with CGP) Images are representative of five independent experiments. The products were analyzed in SDS-PAGE 7.5 %. The gel stained with Coomassie blue. Hydrolytic activity of CGP was inhibited under free calcium conditions and 5 mM EDTA.

Figure 7

Binding of CGP to VE , CE , FE and sperm. Proteins of VE , CE , FE and sperm were resolved on 7.5 % SDS-PAGE and transferred to nitrocellulose. The membrane was incubated with biotin-labeled CGP and reveled with streptavidine peroxidase by chemoluminiscence. Components of CGP bind to gp120, gp41 and gp38 but not to gp75. No evidence of binding to sperm was observed. A, binding of labeled CGP ; B, negative controls; C, PAS-stained proteins. Images are representative of three independent experiments.