LOCATION OF THE PH-20 PROTEIN ON ACROSOME-INTACT AND ACROSOME-REACTEDSPERMATOZOA OF CYNOMOLGUS MACAQUES

Fluorescence microscopy and transmission electron microscopy (TEM) wer e used to determine the location of the membrane protein PH-20 on sper matozoa of cynomolgus macaques. Rabbit antiserum raised against recomb inant cynomolgus macaque sperm PH-20 was used as the primary antibody, and the second antibody was goat anti-rabbit IgG conjugated with eith er fluorescein isothiocyanate or 15 nm gold particles. Spermatozoa wer e evaluated before capacitation and after capacitation and induction o f acrosome reactions with calcium ionophore A23187. In sperm suspensio ns with a high percentage of intact acrosomes, fluorescence labeling w as observed uniformly over most of the sperm head. The sperm midpiece and tail were not labeled. In sperm suspensions with a high percentage of acrosome reactions, most spermatozoa labeled intensely over the an terior sperm head, but labeling of the posterior sperm head was greatl y reduced. TEM of acrosome-intact spermatozoa revealed gold particles distributed uniformly on the plasma membrane overlying the acrosome, t he equatorial segment, and mot of the post-acrosomal region. After the acrosome reaction, gold label was present on the inner acrosomal memb rane and on the plasma membrane overlying the equatorial segment. Very little label was present on the plasma membrane in the post-acrosomal region of acrosome-reacted spermatozoa. The location of PH-20 on the surface of macaque spermatozoa suggests a function for this protein in primary and/or secondary binding to the zona pellucida. The apparent decrease in amount of PH-20 on the posterior head of macaque spermatoz oa following the acrosome reaction is consistent with the migration of this protein to the inner acrosomal membrane, as demonstrated previou sly for the homologous PH-20 protein of guinea pig spermatozoa.