GAMETE BIOLOGY AND GAMETOGENESIS - A
Monday, August 2, 2004
10:30 AM–12:30 PM
(144) CYTOPLASMIC LOCALIZATION DURING TESTICULAR BIOGENESIS OF THE MURINE mRNA FOR SPAM1 (PH-20), A PROTEIN INVOLVED IN ACROSOMAL EXOCYTOSIS.
Martin-DeLeon, Patricia1, Morales, Carlos2, Badran, Haitham2, El-Alfy, Mohamed3, Zhang, Hong2, Men, Hongsheng2, 1 University of Delaware, Newark, DE2 McGill University, Montreal, Quebec, Canada3 University of Laval, Laval, Quebec, Canada
ABSTRACT- Best known for its hyaluronidase activity, the Sperm Adhesion Molecule1 (SPAM1) is the most widely conserved sperm antigen with multiple roles in mammalian fertilization. Earlier we showed that transcripts for the murine Spam1 are compartment-talized in spermatids. The goal of the study was to determine the specific intracellular location of the mRNA and protein as well as the ability of the protein to mediate induction of the acrosome reaction. Light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of Spam1 mRNA in the testis. Transcripts were first detected in step 3 round spermatids, gradually increased until step 8 and abruptly decreased between step 9-11. They were predominantly localized near the ER and were not dispersed throughout the cytoplasm. Immunohistochemistry shows that translation starts 24-48 hr after the initiation of transcription, indicating that the mRNA is transcriptionally regulated, and reveals that the protein is present on both the head and tail of sperm in the seminiferous tubules. Immunocytochemistry confirmed the location of Spam1 on the tail of testicular sperm and demonstrated that it is localized to the principal piece. Spam1 on epididymal sperm is localized to the midpiece of the tail and moves from a uniform distribution on the head in the caput to a regionalized pattern on the anterior and posterior head in caudal sperm. Spam1 on the surface of caudal mouse sperm was shown to mediate the increase in acrosome reactions induced by the synergistic effects of HA and progesterone, as confirmed in sperm from the Rb(6.16) translocation Spam1 mutants. The similar response of human and mouse sperm to these agonists of the acrosome reaction, underscores the usefulness of the mouse as a model to study specific roles of SPAM1 in fertilization events.
KEY WORDS: RNA compartmentalization, acrosome reaction, spermatid, membrane proetin