Amphibian oocytes release HSP70 during spawning: possible role in fertilization.

COUX, GABRIELA; MOUGUELAR, VALERIA S.

Potrero de los Funes

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

SAIB

We recently found evidence suggesting a role for HSP70 proteins(HSP70s) in sperm-oocyte interaction in the amphibian Bufoarenarum. In this work our aim was to study their expression andbehavior during fertilization. We detected HSP70s present in B.arenarum oocyte in the absence of any stress by Western-blot usingtwo different antibodies. 2D electrophoresis experiments detectedtwo isoforms with pIs of 5.25 and 5.45. We studied their subcellulardistribution isolating total membranes, cytosol and plasmamembranes. HSP70s were present in all these fractions. Weconfirmed these results by immunofluorescence microscopy andalso found that HSP70 signal was present in the vitelline envelope(extracellular matrix). To further test this, we performed Westernblot 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 tubulincould not be detected. In vitro fertilization assays in the presence ofanti-HSP70 polyclonal antibodies showed diminished fertilizationscores at low sperm concentrations (105 cells/ml). Humanrecombinant His-tagged HSP70 was able to specifically bind tosperm in vitro. Our results suggest for the first time i) the presence ofHSP70s in the extracellular structures of the oocyte and ii) a role foroocyte HSP70s in fertilization.