Hsp70 and integrin beta 1 in Bufo arenarum oocytes: their role during fertilization.

MOUGUELAR, V. S.; CABADA, M. O.; GABRIELA COUX

Rosario

Congreso; XL Reunion Anual Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular; 2006

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular

Previously we provided evidence on the involvement of HSP70 and a RGDS-sensitive integrin in sperm-oocyte plasma membrane interaction. Our aims were to analyze the effect of the presence of the RGDS peptide and antibodies against HSP70 in the fertilization media and to detect the presence of b1 integrin and its subcellular localization. We performed in vitro fertilization assays with increasing concentration of sperm (104, 5. 104, 105, 3.3 .105 y 106 cells/ml) in the presence of RGDS 1mM, a-Hsp70 antibody (1:16 dilution) and in the absence of both (control). In all the sperm concentrations the control values were higher than in the presence of RGDS or a-Hsp70 antibody. In both cases at 3.3 .105 cells/ml the differences were maximum and significant (in fertilization percentage, mean ±SEM: control= 78.2±4.5, RGDS=43.4±10.9 a-Hsp70=51.5±5.0, n=4, p<0.05). Using differential centrifugation we isolated plasma membranes, microsomes and the cytosolic fraction. Using Western blot with the 8C8 antibody (Hybridoma Bank, against the Xenopus laevis homolog) we detected four major bands of 33, 51, 104 y 133 kDa, corresponding to mature b1 integrin and its precursors in plasma membranes and microsomes. Our results suggest that b1 integrin expresses in B. arenarum oocytes and that both, b1 integrin and Hsp70, are involved in the oocyte-sperm interaction at the plasma membrane level.