Identification of vitelline envelope glycoproteins with sperm binding capacity in Bufo arenarum

BARRERA, DANIEL; MICELI, DORA C.

San Miguel de Tucumán, Tucumán

Congreso; XLV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Vitelline envelope (VE) has an active participation in sperm-oocyte interaction in B. arenarum. It has been shown that one of the key events by which oocytes could be fertilized is related with the proteolytic cleavage of VE glycoproteins (gp) in the first portion of the oviduct. As a consequence of the action of the protease oviductin, the relative amount of 39 kDa gp and 42 kDa gp increase markedly. In the present study, we investigated whether gp39 or gp42 have sperm binding capacity. Sperm were incubated with total VE proteins coupled to biotin. After washes the sperm were lysated and the pool of proteins obtained was separated by SDS-PAGE and electroblotted onto membrane. The biotin-labeled gp were located by staining with streptavidine-peroxidase. As a result, gp39 and gp42 were detected, suggesting that both gp have sperm binding capacity. Subsequently for analyzing thoroughly the role of gp39 we probed the effect of anti-gp39 antibodies on IVF. The fertilization rate significantly decreased when oocytes were pre-incubated with antibodies indicating that gp39 is involved in sperm binding to oocyte. Furthermore, inmunomarcation studies by electronic microscopy showed gp39 placed throughout the thickness of the VE. In conclusion, gp39 and gp42 were probably involved in sperm-oocyte binding.