The biochemistry of the acrosome reaction.

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.

Calcium mobilization and influx during sperm exocytosis.

We have previously shown that two intracellular events which occur during capacitation of bovine sperm are the formation of actin filaments on the plasma and outer acrosomal membranes and the attachment of a PIP2-specific phospholipase C (PLC) to this membrane bound F-actin. This PLC plays an essential role in sperm exocytosis (acrosome reaction). In the present report, we further elucidated the role of this PLC using a PIP2-specific PLC of bacterial origin. This PLC is different from the endogenous sperm PLC in that it is calcium independent and not inhibited by neomycin. Here we report using bovine sperm that this bacterial PLC can restore actin release from extracted membranes as well as membrane fusion in a cell-free assay when the endogenous PLC is inhibited by neomycin. The sperm PLC requires 2 microM calcium for half maximal activation, while half maximal actin release from extracted plasma membranes occurs at 80 microM. Extracted sperm membranes were examined for calcium pumps and channels. Sperm plasma membranes were found to possess a thapsigargin insensitive calcium pump and calcium channels which are opened by phosphorylation by protein kinase C. The acrosomal membrane possesses a calcium pump which is inhibited by thapsigargin and calcium channels which are opened by cAMP. These observations are discussed in terms of a model of acrosomal exocytosis which involves a calcium rise that occurs in two stages resulting from calcium mobilization from internal stores followed by influx of extracellular calcium.

Sperm exocytosis reconstructed in a cell-free system: evidence for the involvement of phospholipase C and actin filaments in membrane fusion.

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.

A 70 kDa protein is transferred from the outer acrosomal to the plasma membrane during capacitation.

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. Furthermore, 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.

Growth inhibition of breast cancer cells induced by exogenous ATP.

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.

A cell free system reveals that capacitation is a prerequisite for membrane fusion during the acrosome reaction.

Plasma and outer 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 the chlorophyll a-N,N'-dioctadecyloxacarbocyanine-p-toluene sulfonate (DCY) method [(1984) Biochim. Biophys. Acta 769, 531-542]. The requirement for capacitation, as well as the effects of pH, calcium and spermine, 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 intracellular pH and calcium concentration, which must precede the acrosomal reaction, are required for the membrane fusion event.

Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity.

The dynein arms that power ciliary motility are normally permanently attached by one end exclusively to subfiber A of each axonemal doublet (N) while the other (head) end transiently attaches to the subfiber B of the adjacent doublet (N + 1) to produce sliding of the doublets. In Tetrahymena axonemes, sliding of contiguous groups of doublets is induced by ATP suggesting that, in the absence of exogenous protease, there may be sets of potentially active and potentially inactive or refractory arms in a single axoneme. In the presence of a non-hydrolyzable analog of ATP, beta,gamma-methylene adenosine 5'-triphosphate (AMP-PCP), about half the doublets in an axonemal preparation retain all arms bound to subfiber A, but half the doublets show long regions where some arms are pulled away from subfiber A of doublet N and attached to subfiber B of doublet N + 1 by their head ends. In AMP-PCP-induced splaying, positional information regarding arm state is retained. Analysis reveals that throughout regions where B subfiber attachment is found, small groups of about four subfiber B attached arms alternate with groups of about four arms that remain attached to subfiber A. This unique pattern of attachment suggests that arms function co-operatively in groups of four. Further, the repetition of the pattern is reminiscent of metachronal activity seen at higher levels of biological organization. This suggests that in these regions we have instantaneously preserved groups of arms capable of attaching to and detaching from doublet N + 1 in rapid succession. This appearance could be used to delineate the potentially active sets of arm, primed for mechanochemical activity, within an axoneme.

Experimental artifacts in the study of mucociliary transport.

Mucociliary transport was studied on frog palate epithelium preparations. Three preparations were used: the detached palate preparation (prepared by detaching the upper part of the head from the frog), the live frog preparation (prepared by exposing the palate of a sedated live frog), and the thin preparation (prepared by excising a 1-mm thin section). Because the transport behavior in each of the 3 preparations is different, extirpation or sedation or excision, or all 3, introduce artifacts into the system.

Stimulation of mucus secretion, ciliary activity, and transport in frog palate epithelium.

Particle transport velocity and ciliary beat frequency, at the level of a single cell of the epithelium, were measured simultaneously. The preparation used keeps the mucociliated epithelium of the frog palate functionally intact but is thin enough for light to be transmitted. The observations confirm that there exists a resting, or unstimulated, state of the epithelium in which the cilia do not beat. It is shown that tactile stimulation (contact with a small 50- to 75-microns foreign particle or with a fine wire probe) restarts ciliary beat. If the epithelium has not been depleted of its mucus, normal ciliary beat frequency is restored, and there is particle transport at the normal velocity. Only the cilia surrounding the moving particle in a patch about 10 times larger are beating at one time. Beat frequency is highest in the center of the patch, near the particle, and tapers to zero toward the edge. Mucus has to be present for particle transport to occur. Particles impacted on a depleted epithelium are not moved. The placement of previously collected endogenous mucus onto a depleted epithelium produces full ciliary activity and normal particle transport. The moving patch of beating cilia corresponds to a plaque of mucus surrounding the particle being transported. This plaque was produced upon first impact of the particle, presumably by mucus secretion, from the epithelial region which then surrounds it. Stimulation of a quiescent nondepleted epithelium with a wire probe induces a normal ciliary beat frequency that gradually decreases to zero. Stimulation by a wire probe of a mucus-depleted epithelium produces a level of initial beat frequency much below normal. Depletion of the epithelial preparation is by an episode of "creeping" over a glass surface. Depletion of the epithelium could be demonstrated histochemically. Analysis of the data of particle velocity and beat frequency is consistent with a wave-length of 45 microns for the metachronous wave.