PURIFICATION OF BOVINE SPERM MEMBRANE-PROTEINS INVOLVED IN SPERM-OOCYTE INTERACTIONS: ADHESION, FUSION, AND ACTIVATION

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At fertilization interactions between sperm and oocyte proteins are pivotal to oocyte activation, fusion and embryo development. The proteins involved in bovine activation have not yet been isolated, identified or characterized. A working hypothesis for the interaction between the sperm and the oocyte is that protein ligands on the inner acrosomal membrane of the sperm bind to protein receptors on the oocyte vitelline membrane. Previously, our laboratory utilized in situ fluorescent labeling of proteins and 2-D gel electrophoresis to identify candidate sperm protein ligands involved in bovine sperm-oocyte interactions. To further this investigation ion exchange chromatography was used to isolate and quantify the proteins of interest derived from the 2-D gel studies. Ion exchange chromatography (IEX) is based on the binding of a charged sample to an oppositely charged compound attached to an insoluble crosslinked beaded agarose matrix. Target protein binding is electrostatic and therefore reversible. Consequently, when a protein is exposed to a pH below its pI it assumes a positive net charge and will bind to a cation exchanger. Briefly, sperm were capacitated and acrosome reacted by incubating approximately 50,000,000 sperm in sperm TL with 6 mg/ml BSA, 0.01 mg/ml heparin, and 100 μg/ml lysophosphatidylcholine at 39°C for 15 min. The acrosome reacted sperm were lysed in buffer containing 8 M urea, 4% CHAPS, 40 mM Tris, and complete protease inhibitor cocktail (Roche Diagnostic, Manheim, Germany) and then applied to a 1 ml HiTrap SP XL column and an AKTA FPLC (GE Healthcare, Piscataway, NJ) automated system at a rate of 1 ml/min. Sequential 0.5 ml fractions were collected and concentrated using vacuum centrifugation to a final volume of approximately 30 μl. The fractions were subjected to SDS-PAGE electrophoresis to identify the fractions containing the protein(s) of interest based on MW and pI. The bands corresponding to the correct MW were analyzed by mass spectrometry and identified by a search of the Mascot database. Densitometric analysis of the gel was also performed using Scion Image software to quantify the specific protein(s) of interest and resulting concentrations were found to range between 0.1–1.0 mg/ml. An additional approach was also used to aid in identifying, isolating, and enriching the proteins involved in sperm-oocyte interactions; the Pierce ProFoundTM Sulfo-SBED Biotin Label Transfer kit was used. Briefly, zona-free oocytes were biotinylated using the NHS ester of the Sulfo- SBED, incubated with capacitated and acrosome reacted sperm, and any interacting proteins were captured by the photoreactive aryl azide moiety. After lysis the biotinylated protein complex was captured utilizing Promega Streptavidin MagneSphere Paramagnetic Particles and identified using three steps: trypsin digestion, mass spectrometry, and database search. Many of the identified proteins currently have no known role in fertilization, but do have many interesting biological functions in other systems including: fusion, signal transduction, cytoskeleton dynamics, and involvement in protein-protein interactions. Work is ongoing to characterize their bioactivity in the bovine fertilization process. (poster)