Amphibian oocytes release HSP70 during spawning: possible role in fertilization

COUX G., MOUGUELAR, V.S.

San Luis

Congreso; Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Celular; 2011

We recently found evidence suggesting a role for HSP70 proteins (HSP70s) in sperm-oocyte interaction in the amphibian Bufo arenarum. In this work our aim was to study their expression and behavior during fertilization. We detected HSP70s present in B.arenarum oocyte in the absence of any stress by Western-blot using two different antibodies. 2D electrophoresis experiments detected two isoforms with pIs of 5.25 and 5.45. We studied their subcellular distribution isolating total membranes, cytosol and plasma membranes. HSP70s were present in all these fractions. We confirmed these results by immunofluorescence microscopy and also found that HSP70 signal was present in the vitelline envelope (extracellular matrix). To further test this, we performed Western blot analysis in isolated vitelline envelopes (crosslinked and not) and in egg water (diffusible material from deposited oocytes). HSP70s were present in these two fractions, while actin or tubulin could not be detected. In vitro fertilization assays in the presence of anti-HSP70 polyclonal antibodies showed diminished fertilization scores at low sperm concentrations (105 cells/ml). Human recombinant His-tagged HSP70 was able to specifically bind to sperm in vitro. Our results suggest for the first time i) the presence of HSP70s in the extracellular structures of the oocyte and ii) a role for oocyte HSP70s in fertilization.