Presence and role of beta1 integrin in the gametes of the amphibian Bufo arenarum

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

Villa Carlos Paz, Córdoba, Argentina

Congreso; XLIV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

We previously found that the b1 integrin expresses in the plasma membrane of B. arenarum oocytes and has a role in the process of fertilization (this was determined by in vitro fertilization assays in the presence of the peptide RGDS). Our aims now were to: i) begin the molecular characterization of B. arenarum b1 integrin, ii) analyze the presence of b1 integrin on the male gamete (sperm) and its role during fertilization.We carried out ovary total RNA isolation, retrotranscription and performed PCR using degenerated primers. The amplified DNA was cloned and sequenced. The obtained 235 bp sequence had strong homology to ?1 integrins (563-639 amino acid sequence). The presence of b1 integrin in sperm was assayed by Western Blot and immunofluorescence using the 8C8 antibody (Hybridoma Bank). These studies revealed that the protein is present predominately in sperm heads. Preliminary in vitro fertilization assays using sperm pre-treated with the RGDS peptide (a known integrin interacting motif) suggest that sperm b1 integrin would not be necessary for the fertilization process to occur (in fertilization percentage, mean + SEM: Control = 56.1 + 2.8; RGDS pre-treated sperm = 59.4 + 6.5, n=3). Our results suggest that although b1 integrin is present in both B. arenarum gametes: sperm heads and oolemma, only plays a role in the oocyte during fertilization.