The binding of the spermatozoon to the oocyte zona pellucida (ZP) occurs via specific receptors localized over the anterior head region of the spermatozoon. Zona pellucida binding stimulates the spermatozoa to undergo the acrosome reaction resulting in the release of hydrolytic enzymes and in the exposure of new membrane domains, both of which are essential for fertilization. We suggest that ZP binds to at least two different receptors in the plasma membrane. One (R) is a Gi-coupled receptor that activates phospholipase C (PLC) beta 1. The other (TK) is a tyrosine kinase receptor coupled to PLC gamma. Binding to R would regulate adenylyl cyclase (AC) leading to elevation of cAMP and protein kinase (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane which releases Ca2+ from the interior of the acrosome to the cytosol. This is the first, relatively small, rise in [Ca2+]i (I) which leads to activation of the PLC gamma. The products of phosphatidyl-inositol bisphosphate (PIP2) hydrolysis by PLC diacylglycerol (DAG) and inositol-trisphosphate (IP3) will lead to PKC translocation to the plasma membrane and its activation. PKC opens a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II) higher increase in [Ca2+]i. The Gi or TK can also activate an Na+/H+ exchanger leading to alkalization of the cytosol. PKC also activates phospholipase A2 (PLA2) to generate arachidonic acid (AA) from membrane phospholipids. AA will be converted to prostaglandins (PG) and leukotriens (LT) by the enzymes cyclooxygenase (COX) and lipoxygenase (LOX) respectively. The increase in [Ca2+]i and pH leads to membrane fusion and acrosomal exocytosis.

Plasma and oukr acrosomal membranes were extracted from bovine spermatozoa and used in an in vitro fusion assay. Fusion was revealed by monitoring the merging of lipids using Ihe chlorophyll &V,N'-dioctadecyloxacarbocyanine-ptolucne sulfonate (DCY) method [(1984) Biochim. Biophys. Acta 769. 531-5421. The requirement for capacitation, as well as the effects of pi-i, calcium and @ermine, on membrane fusion in our cell-free system were similar to those observed in vivo on the acrosomal reaction. This demonstrates for the first time that capacitation and alterations in in~raccllular pH and calcium concentration, which must precede the acrosomal reaction, arc required for the membrane fusion event.

Bull sperm plasma and outer acrosomal membranes were analyzed by SDS-PAGE. Analysis of the plasma membrane proteins revealed the presence of a 70 kDa band the prominence of which is enhanced after capacitation. This protein was found to bind to zona pellucida intact oocytes. PAGE analysis of outer acrosomal membrane proteins also reveals the presence of a 70 kDa band, but its prominence decreases after capacitation. This protein also binds to zona pellucida intact oocytes. Futhermore, the 70 kDa outer acrosomal membrane protein is recognized in Western blot analysis by antibodies to plasma membrane proteins and vice versa. The results indicate that the 70 kDa acrosomal and plasma membrane proteins are the same. This 70 kDa protein would thus be a zona pellucida binding protein which is initially stored in the outer acrosomal membrane and transferred to the plasma membrane during capacitation, enabling it to function in egg-sperm binding.

Addition of ATP (> 0.1 mM) to cultures of human breast cancer T47D cells resulted in an inhibition of cell proliferation. The inhibition was found to be specific for ATP, and dependent on its concentration. Growth inhibition continued for at least three days, although ATP and its hydrolysis products were metabolized within one day. Conditioned medium from ATP-treated cultures (CM+) was found to inhibit the growth of cells that were not exposed to ATP. This is an indication that extracellular factors, besides ATP, are involved in the inhibition process. The inhibition was maintained after dialysis of the CM+, using an 8 kDa cut-off membrane. Conditioned medium from untreated cultures (CM-), however, only slightly affected cell growth. The data suggest that the CM(+)-induced cell growth inhibition is mediated by an ATP-activated growth inhibiting factor. Flow microfluorometry and thymidine incorporation experiments have shown that the growth arrest is mainly due to the elongation of the S-phase of the cell cycle.

We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.