The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: characterization of its most prominent polypeptide (IAM38)

A consequence of the acrosome reaction is to expose the inner acrosomal membrane (IAM), which is a requirement for the sperm's ability to secondarily bind to and then penetrate the zona pellucida (ZP) of the mammalian oocyte. However, the proteins on the IAM responsible for binding and presumably penetrating the zona have not been identified. This issue can be resolved if direct information is made available on the composition of the IAM. For this purpose, we devised a methodology in order to obtain a sperm head fraction consisting solely of the IAM bound to the detergent-resistant perinuclear theca. On the exposed IAM surface of this fraction, we defined an electron dense protein layer that we termed the IAM extracellular coat (IAMC), which was visible on sonicated and acrosome-reacted sperm of several mammalian species. High salt extraction removed the IAMC coincident with the removal of a prominent 38 kDa polypeptide, which we termed IAM38. Antibodies raised against this polypeptide confirmed its presence in the IAMC of intact, sonicated and acrosome-reacted sperm. By immunoscreening of a bovine testicular cDNA library and sequencing the resulting clones, we identified IAM38 as the equivalent of porcine Sp38 [Mori, E., Kashiwabara, S., Baba, T., Inagaki, Y., Mori, T., 1995. Amino acid sequences of porcine Sp38 and proacrosin required for binding to the zona pellucida. Dev. Biol., 168, 575-583], an intra-acrosomal protein with ZP-binding ability, whose precise localization in sperm was unknown. The blockage of IVF at the level of the zona with anti-IAM38 antibodies and the retention of IAM38 after sperm passage through the zona support its involvement in secondary sperm-zona binding. This study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM for identifying other candidates for sperm-zona interactions.