Hsp70 expression and protein tyrosine phosphorylation in Bufo Arenarum oocyte during fertilization

COUX, GABRIELA ; MOUGUELAR, VALERIA S., CABADA, MARCELO O. ;

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

We recently provided evidence for the involvement of HSP70 in sperm-oocyte plasma membrane interaction. On the other hand, although protein tyrosine phosphorylation plays a crucial role in many biological processes, little is known about its participation in fertilization. Our aims were to analyze HSP70 expression in the oocyte and if protein tyrosine phosphorylation is a feature of B. arenarum fertilization. We performed in vitro fertilization assays at the end points 1, 5, and 10 minutes and fractions enriched in oocyte plasma membranes (PM), microsomes, and cytosol were prepared by differential centrifugation. Before and after fertilization, HSP70 expression was assessed by Western blot using a-DnaK antibodies and protein tyrosine phosphorylation by using the PY350 antibody (Santa Cruz Biotech., against phosphotyrosine residues). We found that HSP70 is exclusively detected in PM and that is rapidly down-regulated after fertilization. Protein phosphorylation was found to be increased in cytosol, microsomes, and PM after fertilization. In particular, we detected four bands with increased tyr-phosphorylation in cytosol (approx. 42, 33, 30, and 27 kDa) and only one in PM and microsomes (approx. 42 kDa). Our results suggest that in B. arenarum, fertilization involves changes in protein tyr-phosphorylation and removal of HSP70 from oocyte plasma membranes.