Amphibian Oocytes Release Heat Shock Protein A During Spawning: Evidence for a Role in Fertilization

Abstract

Heat shock proteins A (HSPAs, previously known as HSP70s) are widely distributed proteins originally linked with heat shock but now associated with several normal cellular functions. We recently found indirect evidence suggesting a role for HSPAs in sperm-oocyte interaction in the amphibian B. arenarum. In the present work our aim was to study its expression, sub-cellular distribution and role during fertilization. By western blot using two different antibodies we detected HSPAs present in B. arenarum oocyte in the absence of any stress. We performed 2D electrophoresis and detected two isoforms with pIs of 5.25 and 5.45. We studied its sub-cellular distribution isolating total membranes, cytosol and plasma membranes. HSPAs were present in all these fractions. We confirmed these results by immunofluorescence microscopy and also found that the HSPA signal was present in the vitelline envelope. To further test this, we performed western blot analysis in isolated vitelline envelopes and in egg water (diffusible material from deposited oocytes). HSPAs were present in these two fractions. Moreover, human recombinant his-tagged HSPA (HSPA1A) was able to specifically bind to sperm in vitro (mid-piece) and enhance sperm membrane integrity. In vitro fertilization assays in the presence of anti-HSPA polyclonal antibodies showed diminished fertilization scores at low sperm concentrations (10(5) cells/ml). Our results suggest that HSPAs are present in intra and extracellular structures of non-stressed B. arenarum oocytes, participate in fertilization and that their release during spawning plays a role in sperm membrane integrity.