PARENT SESSION

(M532) INNER ACROSOMAL PROTEIN (IAM32P) FORMS A COMPLEX WITH SP47 AND ACROSIN IN BOAR SPERMATOZOA.

Manandhar, G¹, Barajas-Espinosa, A², Young-Joo, Yi¹, Oko, R², Sutovsky, P^{1, 3}, ¹ University of Missouri-Columbia, Columbia, MO² Queen's University, Kingston, ON, Canada³ University of Missouri-Columbia, Columbia, MO

ABSTRACT- The Inner Acrosomal Membrane (IAM) of sperm heads adjacent to the subacrosomal region of the perinuclear theca is exposed after acrosomal exocytosis forming the leading edge during sperm-zona interaction. In eutherian mammals, the exposed IAM is likely to be involved in secondary binding of acrosome reacted spermatozoa on the zona pellucida and zona penetration. A polyclonal antibody anti-IAM32, prepared and affinity purified against a 32 kDa bull IAM protein recognized 30, 32 and 35 kDa triple bands in Western blots of boar sperm extracts. Immunofluorescence and pre-embedding immunogold electron microscopy (EM) studies showed that the antibody labels the inner acrosomal membrane of acrosome reacted boar spermatozoa. In post-embedding immunogold EM, IAM32 epitope (IAM32p) was found to be located all along the IAM of the intact as well as acrosome reacted bull spermatozoa. Zona penetrated spermatozoa retained IAM32p on the heads but after fusion with oocytes they were not detectable. The presence of antibody in the medium did not affect the rate of pig in vitro fertilization. Immunoprecipitation of the boar sperm extract with the antibody pulled out two additional protein bands of 45 and 47 kDa sizes detectable in coomasssie stained SDS-PAGE. The protein bands were excised separately from the SDS-PAGE and analyzed by MALDI-TOF. Mass fingerprinting and Mascot data search revealed that the 47 kDa band comprised lactadherin (SP47), a 47 kDa milk globulin implicated in sperm-egg interaction. The 45 kDa band matched amino acid sequences of /-acrosin, preproacrosin and acrosin precursors. These observations suggest that putative IAM32p is involved in anchoring SP47 and acrosins on the IAM rather than being directly involved in zona binding or zona penetration in porcine fertilization. Supported by F21C UM-C and USDA-NRI award #2002-02069 to PS.

KEY WORDS: Inner acrosomal membrane, Acrosome reaction, IAM32, MALDI