Reproduction Abstracts

Objective: Considering the recent interest in the use of hyaluronan binding as a sperm selection marker for ICSI, it is becoming increasingly important to revisit and characterise the sperm hyaluronic acid binding proteins (HABPs) that are an integral part of the selection process. To this end, the HABPs in non-capacitated, capacitated and acrosome reacted spermatozoa were investigated.

Design: A laboratory based investigation using immune-cytochemical and biochemical techniques.

Methods: Human and bovine semen samples were processed using density gradient centrifugation (DGC). Prepared spermatozoa were either permitted or not permitted to capacitate and acrosome react in sperm preparation medium (containing or not containing calcium, albumin and NaHCO₃). Samples were subsequently exposed to hyaluronan conjugated with tetramethylrhodamine isothiocyanante (HA-TRITC) and to antibodies to the major HABPs (CD44 and RHAMM). Capacitation and acrosome status, respectively, were monitored using chlorotertacycline (CTC) and the Pisum sativum agglutinin (PSA-FITC). Membrane proteins extracted from capacitated sperm were also probed for HA-binding proteins after SDS PAGE and western blotting.

Results: The results indicated that two major HABPs, CD44 and RHAMM are located on the anterior acrosome and post-acrosomal sheath of non-capacitated bovine spermatozoa. In addition, these receptors became more restricted to the anterior of the acrosome and were essentially lost in capacitated spermatozoa following the acrosome reaction. However, HA-TRITC (a non-target-specific hyaluronan binding reagent) strongly labelled the sperm plasma membrane and the post-acrosomal sheath. HA-TRITC labelling was most intense on the plasma membrane of capacitated spermatozoa and was particularly strong on acrosome reacted sperm. Western blot analysis of SDS-PAGE resolved sperm membrane proteins revealed a number of bands recognized by anti-CD44 and anti-RHAMM antibodies.

Conclusion: The presence of hyaluronic acid binding proteins was confirmed in bovine and human sperm by a HA ligand or by antibodies to CD44 and RHAMM and were shown to be influenced by the cells capacitation and acrosomal status.