WCRB2014 POSTER PRESENTATIONS (1) (335 abstracts)
Detection of SPAM1 in the bovine oviductal fluid
Omar Salvador Acuña 1 , Maria Jimenez-Movilla 1 , Ismael Vilella 1 , Lourdes Jara 1 , Sebastián Cánovas 2 , Pilar Coy 1 , Manuel Avilés 1 & Maria Jose Izquierdo Rico 1
1University of Murcia, Murcia, Spain; 2LARCEL, Sevilla, Spain.
Introduction: The sperm adhesion molecule 1 (SPAM1) is a glycoprotein present in the sperm membrane and it is involved in degradation of cumulus matrix and ZP binding. In the mouse, SPAM1 is secreted by the oviduct and the uterus and is acquired by the sperm during its transit through the genital tract. Our aim was to determine the presence of SPAM1 in the bovine oviduct.
Materials and methods: Ovaries and oviducts were obtained from slaughterhouse cows at the preovulatory stage. PCR analyses were performed using isolated cells from the ampullary mucosa. The oviductal fluids (OF) were obtained by aspiration and analyzed by electrophoresis, immunoprecipitation and western blot (WB). To identify if the SPAM1 is present in the oviductal exosome the Exoquic kit was used. For proteomic analysis, different samples were digested with trypsin and analyzed with Agilent Ion-Trap XCT-Plus mass spectrometer equipped with an electrospray interface. SPAM1 isolated by affinity chromatography was incubated with immature ovarian oocytes and analyzed by confocal microscopy.
Results and discussion: An amplicon of 210 pb that corresponds to the SPAM1 RNAm was obtained and confirmed by automatic sequencing. Four different bands of 75, 60, 35 and 25 kDa were identified by WB. Moreover, proteomic evidences indicates that the SPAM1 protein is a component of the oviductal exosomes. Isolated SPAM1 bound specifically to the ZP of ovarian oocyte. Future research will be necessary to clarify the role of SPAM1 during in vivo fertilization. This work is supported by MINECO (AGL2012-40180-C03-01-02) and the European Commission (FEDER/ERDF).
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ISSN 2052-1472 (online)
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