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Sperm plasma-membrane-associated glutathione S-transferases as gamete recognition molecules

National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India

View larger version (20K): [in a new window]   Fig. 1. Comparison of activity of plasma-membrane-associated GSTs in different cell types. This figure represents GST activities measured using 1 mM CDNB and 1 mM GSH at a 2 minute time point with purified plasma membrane preparations from (a) ventriculocytes, (b) spleenocytes, (c) brain cells, (d) hepatocytes, (e) testicular germ cells and (f) epididymal spermatozoa. The inset shows sperm plasma membrane GST activity with increasing concentrations of CDNB in the presence of 1 mM GSH. The activities of total cellular membranes are also represented.

View larger version (22K): [in a new window]   Fig. 2. Plasma-membrane-associated GSTs on sperm. (A) Autoradiogram of 125I-GSTs purified by GSH affinity chromatography from extracts of the plasma membrane of surface-iodinated sperm in live conditions (lane b). Lane a represents extracts of sperm after removal of the plasma membrane. (B) SDS-PAGE (12%) of GSH affinity-purified GSTs from sperm plasma membrane preparations. Lane a, molecular weight marker; lane b, purified GSTs. (C) Non-reducing SDS-PAGE showing immunoprecipitated proteins from sperm plasma membranes using anti-GST antibodies. (lane a) Preimmune serum; (lane b) anti-GST MuN antibodies; (lane c) anti-GST PiN antibodies; (lane d) migration of GSH affinity-purified sperm plasma membrane GSTs used as a control lane. (D) The RP-HPLC elution profile of sperm plasma membrane GSTs purified by GSH affinity chromatography. Peaks 1 and 2 are GST-Mu, Peak 3 is GST-Pi. (E) Western blots of peaks 1 and 2 represented in D probed with anti-GST M1 antibody (lanes a,b) and anti-GST Pi antibody (lanes c,d), respectively.

View larger version (15K): [in a new window]   Fig. 3. Attachment of GSTs to the sperm plasma membrane evaluated after treatment of the plasma membranes with different reagents. The relative GST activity in 100,000 g supernatants of plasma membranes was measured (using 1 mM CDNB and GSH at a 2 minute time point) after treatment with (3) Pronase, 0.5 mg/ml, 10 minutes, (4) Trypsin, 0.5 mg/ml, 10 minutes, (5) Trypsin, 0.2 mg/ml, 10 minutes, (6) 20 mM HCl, 5 minutes, (7) 1 M NaCl, 15 minutes, (8) 0.1% NP 40, (9) PIPLC, 0.05 units/ml, 20 minutes. (1) The GST activity of the cytosol and (2) the GST activity of the plasma membrane. (Inset) Western blots (probed with anti-GST antibody at 1:1000 dilution). Lane a, total plasma membrane proteins; lane b, supernatant of treatment 7; lane c, GSH affinity-purified GSTs from the supernatant of treatment 7; m, molecular weight marker.

View larger version (18K): [in a new window]   Fig. 4. Binding of sperm plasma membrane and blood cell GSTs to 125I-SZP and of labelled sperm GST-Pi and Mu to SZP. Proteins were blotted onto nitrocellulose membranes and were probed with labelled SZP. (A) Dot blots of (a1) total sperm plasma membrane GSTs probed with 125I-SZP, (a2) total sperm plasma membrane GSTs preadsorbed with cold SZP and subsequently probed with 125I-SZP, (b1) total blood cell GSTs probed with 125I-SZP, (b2) total blood cell GSTs preadsorbed with cold SZP and probed with 125I-SZP, (c1) BSA (5 µg) probed with 125I-SZP and (c2) BSA (5 µg) preadsorbed with cold SZP and subsequently probed with 125I-SZP. (B) Volume quantitation of `A' using a Molecular Dynamics Phosphorimager with Imagequant software package. Groups are as described in A. (C) Binding of radioiodinated GST Pi and GST Mu purified from sperm plasma membranes to SZP coated on to microtitre plates (1 µg/well). Binding of (a) labelled sperm plasma membrane GSTs; (b) labelled sperm plasma membrane GSTs preadsorbed with SZP (10 µg/ml); (c) labelled sperm plasma membrane GST-Mu; (d) labelled sperm plasma membrane GST Mu preadsorbed with SZP (10 µg/ml); (e) labelled sperm plasma membrane GST-Pi; and (f) labelled GST Pi preadsorbed with SZP (10 µg/ml).

View larger version (24K): [in a new window]   Fig. 5. Equilibrium-saturation binding of [125I]-labelled sperm plasma membrane GSTs (*GST) to immobilised SZP. SZP (1 µg/well) was immobilised on microtitre plates and [125 I]-labelled sperm plasma membrane GSTs were allowed to bind under different conditions. (a) Moles of total sperm plasma membrane GSTs bound to SZP; (b) specific binding of GSTs, which equals total binding (a) minus non-specific binding (c); (c) moles of total sperm plasma membrane GSTs bound to SZP after blocking with 100 mM cold GST. Data are means±s.e.m.

View larger version (30K): [in a new window]   Fig. 6. Binding of SZP to MuN and PiN peptides immobilised on microtitre plates. Different concentrations of GST PiN and MuN peptides were coated onto microtitre plates and probed with 50 ng of 125I-SZP under different conditions. Competition was carried out with unlabelled SZP or peptides. (A) Saturable binding of 125I-SZP to MuN peptide; (a) MuN peptide + 125I-SZP; (b) MuN peptide + 125I-SZP + excess unlabelled SZP (200 ng); (c) MuN peptide + 125I-SZP + PiN peptide (in solution with 125I-SZP); (d) MuC peptide + 125I-SZP. (B) Saturable binding with 125I-SZP to PiN peptide; (a) PiN peptide + 125I SZP; (b) PiN peptide + 125I-SZP + excess unlabelled SZP (200 ng); (c) PiN peptide + 125I-SZP + MuN peptide (in solution with 125I-SZP); (d) PiC peptide + 125I-SZP. (C) Inhibition profile of binding of 600 nM *GST to SZP (1 µg) in the presence of excess peptides. (D) CD spectra of PiN and MuN peptides. CD spectra of the Pi-N peptide (a) has a characteristic minima of polyproline structure II at 206 nm, that shows that majority of the PiN peptide assumes an extended conformation. MuN peptide (b) has a minima below 200 nm and shows a typical pattern of random coil. *GST, radiolabelled GSTs.

View larger version (30K): [in a new window]   Fig. 7. Binding of biotinylated sperm GSTs to components of goat ZP. Glycoproteins of goat ZP on SDS-PAGE under reducing conditions separate into three bands (lane a). Lane b shows the binding of biotinylated sperm plasma membrane GSTs to band three of goat ZP. Lane c shows the binding of monoclonal antibody MA 451, raised against a ZP 3 peptide from a conserved region, to band three of goat ZP. m, marker.

View larger version (75K): [in a new window]   Fig. 8. Binding of GST Mu and Pi peptides to oocyte ZP. Oocytes were incubated with GST PiN and MuN peptides (10 µg/ml) for 30 minutes at 37°C followed by treatment with rabbit anti-GST PiN and MuN antibodies (1:100) and subsequently stained with anti-rabbit IgG (1:200) conjugated to FITC. This figure shows fluorescence photomicrograph of oocytes incubated with (A) total sperm plasma membrane GSTs. (B) GST PiN peptide. (C) GST MuN peptide. (D) Four-cell embryo incubated with total plasma membrane GSTs. (E-H) Phase-contrast photomicrographs of A-D, respectively. (I) A control oocyte treated with unrelated peptide (tritrypticin-VRRFPWWWPFLRR). (J) Phase contrast of I. Inner cytoplasmic masses (i) have been pushed out to rule out any background staining. Z, ZP. Bars, 100 µm.

View larger version (37K): [in a new window]   Fig. 9. Acrosomal status of sperm undergoing GST aggregation. To visualise aggregation of sperm-surface GSTs, capacitated sperm were incubated with SZP (10 µg/ml/107 sperm) at 37°C, and aliquots of sperm were fixed at different time points in 4% buffered paraformaldehyde. The movement of the GST molecules was visualised by staining with FITC-labelled secondary antibody (1:500). Pisum sativum agglutinin (PSA) conjugated to rhodamine (1:100) was used to stain the same samples to visualise the status of the acrosome. (A) Aggregation of sperm GST Pi on goat sperm surface after treatment with SZP at a 60 minute time point. B shows the same spermatozoa as in microphotograph A stained with rhodamine-labelled Pisum sativum agglutinin showing the intactness of the entire acrosome at 1 hour. (C) Aggregation of sperm GST Mu on goat sperm surface after treatment with SZP at a 60 minute time point. (D) The same spermatozoa as in microphotograph C stained with rhodamine-labelled Pisum sativum agglutinin showing the intactness of the entire acrosome at 1 hour. Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 10. Antibody-induced aggregation of sperm plasma membrane GSTs. Capacitated sperm were exposed to anti-GST antibodies and fixed at different time points in 4% buffered formaldehyde and immunostained using anti-GST Pi antibodies. (A) 0 minutes, (B) 15 minutes, (C) 30 minutes and (D) 60 minutes incubation of live spermatozoa with anti GST PiN antibodies (1:25) at 37°C. (E) The effect of antibody crosslinking of sperm plasma membrane GSTs on acrosin release. Acrosin release was measured after aggregation of GSTs on capacitated spermatozoa and was induced by anti-GST PiN antibodies. The bar shows mu/minute of acrosin released/106 sperm. Capacitated sperm were treated with (a) preimmune serum (1:25), (b) SZP (10 µg/ml), (c) anti-GST-Pi (1:10), (d) anti-GST-Pi (1:25), (e) anti-GST-Pi (1:50), (f) anti-GST-Pi (1:50) + antirabbit IgG (1:100), (g) anti-GSTMu (1:10), (h), Anti-GSTMu (1:25), (i) anti-GSTMu (1:50), and (j) anti-GSTMu (1:50) + antirabbit IgG (1:100). Anti-rabbit IgG (1:50) did not have any effect on acrosin release (data not shown). a versus b,c,f,g,j; e versus f; i versus j: P<0.0001. a versus d: P<0.005. Bar, 20 µm.

View larger version (13K): [in a new window]   Fig. 11. Intracellular Ca2+ concentration of sperm after SZP-induced acrosome reaction. Capacitated spermatozoa (CSP) (107) were incubated with 1 µM of Ca2+-binding dye Fura 2-AM at 37°C for 40 minutes and was incubated with SZP. The fluorescence pattern was subsequently measured. (A) a, CSP + SZP; b, CSP + anti-PiN adsorbed with PiN + SZP; c, (CSP + anti-PiN) + SZP; d, (CSP + anti-MuN) + SZP; e, CSP + SZP adsorbed with PiN; f, CSP + SZP adsorbed with MuN. (B) For microscopic estimation, the number of Fura 2-AM-positive sperm was scored under a Nikon Optiphot fluorescence microscope. a, CSP, b, CSP + SZP, c, (CSP + anti-PiN) + SZP, d, (CSP + anti-MuN) + SZP.