BIOLOGY OF REPRODUCTION 82, 572–579 (2010)Published online before print 30 September 2009.DOI 10.1095/biolreprod.109.076695Hamster Sperm-Associated Alpha-L-Fucosidase Functions During FertilizationJennifer J. Venditti,1Jennifer M. Swann, and Barry S. BeanDepartment of Biological Sciences, Lehigh University, Bethlehem, PennsylvaniaABSTRACTSperm-associated alpha-L-fucosidases have been identified indiverse organisms. Their wide phylogenetic distribution andknown properties support the likelihood thatL-fucose and alpha-L-fucosidase have fundamental function(s) during gamete inter-action. This is consistent with the substantial evidence in theliterature documenting the importance of carbohydrate moietiesduring fertilization. Direct enzyme assays were employed toevaluate the functional distribution of alpha-L-fucosidase inpreparations of hamster sperm. In vitro fertilization wasperformed using Syrian hamster sperm and eggs to identify thefunctional role of hamster sperm-associated alpha-L-fucosidaseduring zona pellucida binding/penetration, sperm-egg mem-brane fusion, and postfusion events. Results reported heredocument the presence of hamster sperm-associated alpha-L-fucosidase and demonstrate that it functions during fertilizationat the stage of sperm-oocyte membrane interaction and/orpostfusion events within the zygote. Understanding the role ofalpha-L-fucosidase during human fertilization could lead todevelopment of improved infertility treatments.DFJ, fertilization, fucose, gamete biology, in vitro fertilizationINTRODUCTIONSuccessful fertilization requires the coordination of species-signature events between the sperm and oocyte. It has beenwell documented that fertilization is a carbohydrate-mediatedevent. Previous studies support roles for carbohydrates insperm-oviduct adhesion [1–4], sperm-oocyte interaction [5, 6],and embryo implantation [7, 8]. Gamete recognition andadhesion on the molecular level involve carbohydrates [9, 10].Substantial evidence in the literature points toward theimportance ofL-fucose. The deoxyhexose L-fucose is acommon terminal residue of both N-orO-linked glycolipidsand glycoproteins [11]. Addition of this residue to moleculescan enable unique functional properties. Its physiological rolesin mammals have been well characterized and reviewed indetail [11]. Apart from being essential to many biologicalfunctions,L-fucose has also been reported to be involvedduring fertilization and has been identified as a component ofgametes [12–16].Fucose is a common component of the glycan chains ofglycoproteins and glycoplipids, and there is a large family offucosyltransferases available to recognize various substratesand add fucose residues selectively. By contrast, mostorganisms have only one or two genes for a-L-fucosidasesthat recognize and cleave terminal fucose residues [17].Considering the known relevance of carbohydrate structuresin reproduction and the common occurrence of fucose residuesat or near the terminal positions in glycans, fucosidases arelikely suspects for key roles in reproduction.For humans in particular, there are distinctive isoforms of a-L-fucosidase that occur in the seminal plasma versus the spermmembrane-associated alpha-L-fucosidase (SMALF) [18–20].The distribution of SMALF within human sperm is unusual,suggesting that it is packaged for actions late during sperm-oocyte interaction. For human sperm, SMALF is crypticallyheld within sperm cells, with a crypticity ratio averaging 3.7 forpermeabilized versus untreated sperm cells [21]. Furthermore,SMALF is enriched within the equatorial segment, positionedfor engagement in the intimate membrane interactions betweensperm and oocyte [21]. Human SMALF is notably stabilized byits in situ membrane association and remains actively stablewithin the timeline, consistent with a delayed fertilization event[22].Mammalian fucosidases have been characterized andreviewed at length [23, 24]. In addition to the common a-L-fucosidases, sperm-associated isoforms of this enzyme havebeen documented in humans [18–20], rats [25–27], Drosophila[28], ascidians [29], some Percidae fishes [30], Unioelongatulus [31], chimpanzees [32], bulls [33], and Syrianhamsters (described in the present study). The presence ofsperm a-L-fucosidase isoforms in this diverse group oforganisms is consistent with the importance of carbohydratesduring fertilization. Several studies provide evidence support-ing roles for fucose containing glycans and/or fucosidasesduring fertilization [29, 34–37].Because direct intervention with human fertilization isundesirable, we have chosen the Syrian hamster for furtheranalysis of the roles of fucosidase during fertilization. Thisrodent provided numerous advantages for such an investiga-tion, allowing testing of multiple hypotheses on the mecha-nisms of gamete interaction and postfusion events.The primary objectives of this study were to assess thepresence of a-L-fucosidase in hamster sperm and oocytes andto evaluate the roles of a-L-fucosidase during fertilization.Direct enzyme assays were performed to measure a-L-fucosidase activity in pellet and supernatant fractions of cauda,capacitated, and acrosome-induced hamster sperm prepara-tions. In vitro fertilization (IVF) was performed to evaluate therole of sperm-associated a-L-fucosidase during 1) zonapellucida (ZP) binding and penetration, 2) sperm-oocytemembrane interaction, and 3) oocyte activation coupled withearly embryogenesis.To evaluate the functional role of a-L-fucosidase duringfertilization, we blocked its enzyme activity using thecommercially available chemical deoxyfuconojirimycin(DFJ). DFJ is a known, ‘‘potent, specific, and competitiveinhibitor’’ of the a-L-fucosidase enzyme [38], and it retains thatdistinction [39, 40]. The chemical structure of this deoxyazo-sugar resembles the natural substrates for a-L-fucosidase.Previous studies in our lab using human sperm have confirmed1Correspondence and current address: Jennifer J. Venditti, Departmentof Biology, Kutztown University, Kutztown, PA 19530.FAX: 610 758 4004; e-mail: [email protected]: 6 February 2009.First decision: 4 March 2009.Accepted: 16 September 2009.Ó 2010 by the Society for the Study of Reproduction, Inc.eISSN: 1529-7268 http://www.biolreprod.orgISSN: 0006-3363572Downloaded from www.biolreprod.org.and documented the specificity of DFJ for fucosidases fromseveral sources [21, 22, 30], including the sperm-associatedisoforms [21]. Results reported