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a Hormones/Growth/Development Group, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9
b Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
c Department of Obstetrics and Gynecology, Division of Reproductive Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
d Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.
1 This work was supported by CIHR (grant 10366) and the Rockefeller Foundation, both awarded to N.T. W.W. and A.A. are awardees of a scholarship from the National Science and Technology Department Agency of Thailand and Thailand Research Funds, respectively. D.M. was awarded an NSERC summer student scholarship (2000, 2001).
2 Correspondence: Nongnuj Tanphaichitr, Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365; ntanphaichitr{at}ohri.ca
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