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Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization

¹ Signalling Programme, The Babraham Institute, Cambridge CB2 4AT, UK
² Institut fur Humangenetik, Universitat Gottingen, D-37073, Germany

View larger version (33K): [in a new window]   Fig. 1. 125I-mZP2 and [3H]suramin bind less efficiently to proacrosin (-/-) than (+/+) mouse sperm. Binding of (A) purified 125I-mZP2 and (B) [3H]suramin to proacrosin (+/+) and (-/-) sperm was detected by autoradiography. Micrographs (top) illustrate the typical distribution of silver grains over the sperm head. Data below show the mean number ± s.e.m. of silver grains over the head region of 100 sperm from each of nine proacrosin (-/-) and nine wild-type (+/+) mice in A, and three acrosin (-/-) and four wild-type (+/+) mice in B. Differences between (+/+) and (-/-) sperm were significant (P<0.01) in both A and B.

View larger version (12K): [in a new window]   Fig. 2. Effects of ionic strength and pH on binding of 125I-ZP glycoproteins to expressed proacrosin peptide. The relative uptake of unfractionated 125I-ZP glycoproteins to expressed proacrosin peptide (expressed as c.p.m. bound, see Fig. 3) was measured in the presence of (A) 0-400 mM NaCl or (B) pH 3.2-8.0 at 100 mM NaCl.

View larger version (43K): [in a new window]   Fig. 3. Relative binding of 125I-mZP2 and 125I-mZP3 glycoproteins to bacterial expressed mouse proacrosin peptide. (A) Coomassie Blue stained proteins separated by reducing SDS-PAGE after extraction from inclusion bodies. Lane 1, pre-induction; lane 2, post-induction showing expression of a major 32 kDa peptide (arrow). (B) Binding of unfractionated 125I-mZP glycoproteins to expressed 32 kDa peptide on western blots. Lane 3, pre-induced bacterial proteins probed with 125I-mZP glycoproteins; lane 4, post-induction showing binding of 125I-mZP glycoproteins to the 32 kDa peptide. (C) Binding of purified 125I-mZP2 (lane 5) and purified 125I-mZP3 (lane 6) to the 32 kDa peptide. (D) Immunological identification of the 32 kDa peptide as expressed residues 38-288 of proacrosin. Lane 7, immune serum. Lane 8, pre-immune serum. (E) Autoradiograph of purified 125I-mZP glycoproteins separated by SDS-PAGE. Lane 9, unfractionated 125I-mZP glycoproteins. Lane 10, purified 125I-mZP2 equivalent to 120 kDa. Lane 11, purified 125I-mZP3 equivalent to 83 kDa. (F) Relative binding of purified 125I-mZP2 and 125I-mZP3 to expressed proacrosin 32 kDa peptide. All experiments were repeated at least 3 times and results shown are representative of the data as a whole. See text for details.

View larger version (35K): [in a new window]   Fig. 4. Effects of sulphated versus non-sulphated competitors on binding of 125I-mZP2 to expressed proacrosin peptide. (A) Typical autoradiograph of a western blot containing the expressed 32 kDa peptide probed with 125I-mZP2 in the presence of a variety of competitors (all at 1 mg/ml). Suramin, dextran sulphate and fucoidan are all effective competitors whereas dextran and soybean trypson inhibitor (SBTI) are ineffective. (B) Relative efficacy of different competitors in preventing binding of 125I-mZP2 to the 32 kDa peptide. Binding is expressed as a percentage relative to controls containing no competitor (taken as 100%). Results are means ± s.e.m. of 3-4 separate experiments.

View larger version (22K): [in a new window]   Fig. 5. Chemical structure of suramin and two of it analogues, NF062 and NF036. Note that suramin is a chemically symmetrical molecule containing three sulphonate groups on each of the terminal naphthalene rings. NF062 on the other hand carries only one terminal sulphonate group whilst NF036 is a truncated asymmetric molecule; see Jentsch et al. (Jentsch et al., 1987).

View larger version (12K): [in a new window]   Fig. 6. Effects of sulphated and non-sulphated competitors on binding of 125I-mZP2 to native mouse proacrosin in sperm heads as detected by autoradiography. Binding is expressed relative to controls containing no competitor (taken as 100%). Competitors were tested at 1 mg/ml.

View larger version (63K): [in a new window]   Fig. 7. Displacement of bound sperm from the ZP in the presence of suphated and non-sulphated competitors (‘pulse-chase’ experiments). (A) Representative micrographs of (+/+) sperm bound to the ZP in (i) control eggs, (ii) eggs after 15 minutes incubation with 1 mg/ml fucoidan, (iii) eggs after 15 minutes incubation with 1 mg/ml suramin. Scale bar, 50 µm. (B,C) Representative results showing the mean number of sperm bound to the ZP during the 35 minutes chase period in the presence of various competitors as indicated, expressed as a percentage of parallel controls at each time point. Zero minutes is taken as 100%. All compounds were tested at 1 mg/ml and experiments repeated at least 3 times.

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