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First published online 5 December 2006
doi: 10.1242/jcs.03258

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Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

¹ Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China
² Department of Obstetrics and Gynaecology, University Central Hospital, 00029 HUS Helsinki, Finland
³ Department of Clinical Chemistry, University Central Hospital, 00029 HUS Helsinki, Finland

View larger version (40K): [in this window] [in a new window]   Fig. 1. Isolation of glycodelin-A binding human sperm surface proteins. (A) Silver staining of 12% SDS-PAGE gel. (B) Detection of biotinylated protein using streptavidin-alkaline phosphatase conjugate. (C) Detection of FUT5 using the polyclonal anti-FUT5 antibody, N-18. Lane 1: sulfo-SBED-conjugated glycodelin-A in non-reducing conditions. Lane 2: sulfo-SBED-conjugated glycodelin-A in reducing conditions. Lane 3: immunoaffinity-purified cross-linked glycodelin-sperm protein complex in non-reducing conditions. Lane 4: immunoaffinity-purified cross-linked glycodelin-sperm protein complex in reducing conditions. Lane 5: similar to lane 4 with the use of human fibroblasts instead of spermatozoa. Lane 6: similar to lane 5 with the use of human fibroblasts instead of spermatozoa. Lane 7: molecular mass marker.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Western blot analysis of sperm protein extract probed with anti-fucosyltransferase antibodies (1:200). (A) D-17 against the C-terminus of FUT5. N-16(B) N-18 (C) and G16 (D) against the N-terminus of FUT3/FUT6, FUT5 and FUT6, respectively. Lane 1: molecular mass marker. Lane 2: 10 µg sperm protein extracts. Lanes 3-5: 5 µg of recombinant FUT3, FUT5 and FUT6, respectively.

View larger version (109K): [in this window] [in a new window]   Fig. 3. Immunolocalization of fucosyltransferase in human spermatozoa with or without Triton X-100 permeabilization. Spermatozoa were incubated with different polyclonal anti-FUT antibodies: D-17 (A), N-16 (B), N-18 (C) and G-16 (D). They were visualized using FITC-conjugated mouse anti-goat IgG.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Competition binding of spermatozoa to saturated concentration (300 pmol/ml) of 125I-glycodelin-A in the presence of glycodelin (n=3) (A) and fucosyltransferase acceptors (n=3) (B).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Purification of sperm FUT. (A) Representative Mono-Q chromatogram of sperm extracts. The protein fractions (1-10) were desalted and further purified using a GDP-agarose column. They were then subjected to 12% SDS-PAGE analysis with silver staining (B) and western blot analysis using the anti-FUT5 antibody N-18 (B).

View larger version (57K): [in this window] [in a new window]   Fig. 6. Co-immunoprecipitation of FUT with glycodelin-A or deglycosylated glycodelin-A. (A) Glycodelin-A. Lane 1: marker. Lane 2: 5 µg FUT3 + 30 pmol/ml glycodelin-A. Lane 3: 5 µg FUT5 + 30 pmol/ml glycodelin-A. Lane 4: 5 µg FUT6 + 30 pmol/ml glycodelin-A. (B) Deglycosylated glycodelin-A. Lane 1: marker. Lane 2: 5 µg FUT3 + 30 pmol/ml deglycosylated glycodelin-A. Lane 3: 5 µg FUT5 + 30 pmol/ml deglycosylated glycodelin-A. Lane 4: 5 µg FUT6 + 30 pmol/ml deglycosylated glycodelin-A. Lane 5: 5 µg sperm FUT + 30 pmol/ml deglycosylated glycodelin-A.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Co-immunoprecipitation of sperm FUT with glycodelin-A in the presence of zona pellucida glycoproteins. The amount of zona pellucida glycoprotein used is expressed as the number of zona pellucida solubilized per microlitre of the final incubation mixture (ZP/µl). Lane 1: 5 µg sperm FUT + 30 pmol/ml glycodelin-A. Lane 2: 5 µg sperm FUT + 30 pmol/ml glycodelin-A + 0.02 ZP/µl. Lane 3: 5 µg sperm FUT + 30 pmol/ml glycodelin-A + 0.1 ZP/µl.

View larger version (21K): [in this window] [in a new window]   Fig. 8. Effect of anti-FUT5 antibody and FUT acceptors on zona pellucida binding of spermatozoa. Spermatozoa was incubated in EBSS/BSA (control) and in (A) EBSS/BSA containing 0.1, 1, 5, 10 and 20 µg/ml anti-FUT antibody D-17 with or without blocking peptide preabsorption, and (B) EBSS/BSA containing FUT acceptors at concentrations of 30, 300 and 3000 pmol/ml. Each point represents the mean of the results of five hemizona binding assays using five zona pellucida and five different sperm samples. *P<0.05, when compared with the control without any treatment.

View larger version (70K): [in this window] [in a new window]   Fig. 9. Binding of sperm FUT5 to intact human zona pellcuida. Human oocytes were incubated with (A) 150 pmol/ml Alexa-Fluor 488 sperm FUT5, (B) 150 pmol/ml Alexa-Fluor 488 sperm FUT5 + 50-fold excess of unlabeled sperm FUT5, or (C) 150 pmol/ml Alexa-Fluor 488 ovalbumin. The results shown are representative of two replicate experiments.

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