Zona pellucida-mediated acrosomal exocytosis in mouse spermatozoa: characterization of an intermediate stage prior to the completion of the acrosome reaction.
The zona pellucida (ZP)-induced acrosome reaction in mouse sperm proceeds in two steps, identified by three sperm fluorescence patterns observed sequentially with the fluorescent probe chlortetracycline. Capacitated, acrosome-intact sperm displaying a B pattern proceed to an intermediate S pattern, and then progress from the S pattern to the fully acrosome-reacted AR pattern. Previously, it was not feasible to characterize the nature of the transient intermediate S pattern. Recently, it was demonstrated that sperm bind to the ZP of eggs treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and undergo a B to S transition, but do not complete the acrosome reaction. These cells accumulate in the S pattern and fail to undergo the S to AR transition (Endo, Y., Schultz, R. M., and Kopf, G. S. 1987a. Dev. Biol. 119, 119-209). The present study utilized ZP from TPA-treated eggs to assess the state of S pattern sperm. The kinetics of the B to S transition of sperm incubated with either structurally intact or solubilized ZP from untreated or TPA-treated eggs are identical. Addition of either solubilized ZP from untreated eggs or A-23187 to S pattern sperm bound to intact or solubilized ZP from TPA-treated eggs induces the S to AR transition, while ZP from TPA-treated or fertilized eggs does not. Loss of the transmembrane pH gradient in the anterior portion of the sperm head, monitored by the fluorescent pH probe 9-N-dodecyl aminoacridine, follows the B to S transition in sperm incubated with ZP from unfertilized eggs, but no loss is observed when the B to S transition is induced using ZP from TPA-treated eggs. Subsequent addition of solubilized ZP from untreated eggs or A-23187 results in the loss of the transmembrane pH gradient of these S pattern sperm. Addition of nigericin to S pattern sperm bound to ZP from TPA-treated eggs discharges the transmembrane pH gradient and causes the S to AR transition. In contrast, nigericin added to B pattern sperm discharges the pH gradient but does not induce a B to S transition. Electron microscopic evaluation of S pattern-arrested sperm using ZP from TPA-treated eggs reveals intact plasma and outer acrosomal membranes. These results suggest that ZP from TPA-treated and fertilized eggs are modified such that the ZP ligands inducing the S to AR transition are lost or are inactivated.(ABSTRACT TRUNCATED AT 400 WORDS)
Kligman
I
I
Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia 19104-6080.
Glassner
M
M
Storey
B T
BT
Kopf
G S
GS
D000177:Acrosome / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002751:Chlortetracycline; D005089:Exocytosis; D005260:Female; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D008854:Microscopy, Electron; D009550:Nigericin / Q000494:pharmacology; D051381:Rats; D013075:Sperm Capacitation; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D015044:Zona Pellucida / Q000502:physiology*
D000001:Calcimycin; D013755:Tetradecanoylphorbol Acetate; D009550:Nigericin; D002751:Chlortetracycline