Submission Number: GEO-4-2-8

Abstract Number: 257

POST-TRANSLATIONAL MODIFICATION OF MOUSE SPERM ACROSOMAL PROTEIN sp56 DURING SPERMATOGENESIS, EPIDIDYMAL MATURATION, AND SPONTANEOUS ACROSOMAL EXOCYTOSIS.

George L Gerton*, Moonchan Cha and Kye-Seong Kim

Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia, PA, USA. ¹

Abstract:
Mouse sperm protein sp56 is a zona pellucida-binding protein with a reported molecular weight (M_r) of 56,000 [Cheng et al. J.Cell Biol. 125:867, 1994]. Our laboratory demonstrated that sp56 is an integral component of the acrosome and that AM67, the guinea pig homologue of sp56, is a 67,000 M_r acrosomal matrix glycoprotein [Foster et al. J. Biol. Chem. 272:12714-12722, 1997]. To resolve the differences in molecular weights between the two species and to examine sp56 during mouse germ cell development and maturation, we examined sp56 from spermatogenic cells and cauda epididymal sperm as well as epididymal sperm that had undergone acrosomal exocytosis during the course of in vitro capacitation. Two affinity-purified polyclonal anti-peptide antibodies were utilized. One, anti-CPT, recognized a central peptide in sp56; the other antibody, anti-VYK, was specific for the C-terminus. By indirect immunofluorescence, sp56 was present within developing acrosomes of spermatids. By western blotting, both antibodies detected maturation-dependent processing of sp56: a doublet of 79,600 and 67,600 M_r in round spermatids, a single band of 67,600 M_r in condensing spermatids, and a 62,100 M_r band in cauda epididymal sperm. During a 1-hour time course of capacitation in vitro, many epididymal sperm underwent spontaneous acrosomal exocytosis accompanied by the release of a 43,200 M_r form of sp56. The processing was apparently due to the proteolytic removal of a C-terminal fragment from sp56 since anti-VYK no longer detected the band observed by anti-CPT. Unprocessed sp56 was not released into the supernatant during the course of capacitation, suggesting that proteolytic cleavage of sp56 is required for its release from the sperm during acrosomal exocytosis. These results are consistent with our alternative paradigm for acrosomal dynamics that proposes a role for acrosomal matrix components in sperm-zona pellucida interactions. Supported in part by NIH HD-22899 to GLG.

Keywords: sperm, acrosome, capacitation, spermatogenesis, acrosomal exocytosis

This abstract is being presented at: 2:45 PM in session:
SESSION 13: CAPACITATION AND ACROSOME REACTION / SPERM MOTILITY