Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.

ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization.

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.

Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction.

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.

Nature of the mouse egg's receptor for sperm.

The zona pellucida is an extracellular coat that surrounds all mammalian eggs. Sperm must penetrate the zona pellucida in order to reach and fuse with the plasma membrane of unfertilized eggs. Penetration is accomplished by a sequence of events involving both egg and sperm. First, sperm must bind to the outer margin of the zona pellucida. Such binding is mediated in a relatively species-specific manner by "sperm receptors" in the zona pellucida. Second, sperm must undergo the "acrosome reaction", a membrane fusion event, in order to traverse the zona pellucida. Here we review results from our own laboratory which demonstrate that, during the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 serves as both receptor for sperm and inducer of the acrosome reaction. Furthermore, we review evidence from our laboratory indicating that the sperm receptor activity of ZP3 is dependent only on its 0-linked carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide portion of ZP3 as well.

Sequential focal and global elevations of sperm intracellular Ca2+ are initiated by the zona pellucida during acrosomal exocytosis.

The acrosome reaction is a Cai(2+)-dependent secretory event that is initiated in mammalian sperm by ZP3, the stimulatory agonist in the egg's zona pellucida. Video image processing-enhanced fluorescence microscopy was used to monitor sperm Cai2+ responses to agonist during exocytosis. Two types of agonist-dependent Cai2+ transport pathways were defined. The first mediates small, transient Cai2+ elevations that are restricted to the sperm head. Dye emission and quenching studies indicate that this focal channel is not voltage-regulated, conducts several divalent metal cations (Co2+, Mn2+, and Ni2+) in addition to Ca2+, and has the properties of a poorly selective cation channel. The second transporter mediates sustained Cai2+ elevations throughout the cell and is pharmacologically identical to the L-type of voltage-sensitive Ca2+ channel. These channels are distinguished by inhibitor sensitivity and by their regulation during sperm maturation. A model is proposed in which ZP3 initiates a G protein-independent opening of the cation channel, producing depolarization of sperm membrane potential and consequent opening of L channels. The coordinate regulation of sperm Ca2+ channels by ZP3 during gamete adhesion promotes acrosomal exocytosis.

Inhibition of in vitro fertilization of mouse eggs: 3-quinuclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa.

The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.

O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity.

Previously, we reported that ZP3, one of three different glycoproteins present in the mouse egg's zona pellucida, serves as a sperm receptor. Furthermore, small glycopeptides derived from egg ZP3 retain full sperm receptor activity, suggesting a role for carbohydrate, rather than polypeptide chain in receptor function. Here, we report that removal of O-linked oligosaccharides from ZP3 destroys its sperm receptor activity, whereas removal of N-linked oligosaccharides has no effect. A specific size class of O-linked oligosaccharides, recovered following mild alkaline hydrolysis and reduction of ZP3, is shown to possess sperm receptor activity and to bind to sperm. The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3.

The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro.

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The t1/2 of this process was 5-10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an ED50 and a maximally effective dosage of 20 and 50 ng protein/microliter, respectively. Zonae pellucidae caused up to a 48-fold increase in the apparent initial rate and a 3- to 4-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida.

Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements.

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion.

Membrane potential (VM) was investigated in mouse and bovine sperm populations. VM was determined from the fluorescence emission of the lipophilic anion, bis(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)), and from the lipophilic cation, 3,3'-dipropylthiodicarbocyanine iodide (DiSC3(5)). Fluorescent signals were corrected for contributions of mitochondrial potentials and apparent VM values were obtained by calibrations in sperm selectively permeabilized with valinomycin or with gramicidin D. The calculated VM values of uncapacitated mouse and bovine sperm were approximately -35 and -30 mV, respectively. In contrast, capacitated populations of mouse and bovine sperm have VM values of -50 to -60 mV. Membrane hyperpolarization is due in part to an enhanced K+ permeability. The development of zona pellucida-activated signal transducing mechanisms during capacitation is dependent upon hyperpolarization. It is suggested that VM alterations regulate the activation state of sperm, thereby suppressing premature acrosome reactions in uncapacitated sperm and permitting capacitated sperm to respond to zona pellucida stimuli.

pH regulation in mouse sperm: identification of Na(+)-, Cl(-)-, and HCO3(-)-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation.

Intracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHi in these cells are not understood. The pHi of mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na(+)-dependent Cl(-)-HCO3- exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates in N-methyl-D-glucamine+ Cl- or in 0.25 M sucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na(+)-H+ antiport pathway and the Na(+)-independent Cl(-)-HCO3- exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHi increases (from 6.54 +/- 0.08 to 6.73 +/- 0.09) that require a functional Na(+)-, Cl(-)-, and HCO3(-)-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHi during capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHi regulator.

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation.

A T-type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the egg's extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole-cell patch-clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage-dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage-dependent facilitation of the current. These data are consistent with the presence of a two-state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.

Caltrin, the calcium transport regulatory peptide of spermatozoa, modulates acrosomal exocytosis in response to the egg's zona pellucida.

Acrosomal exocytosis is initiated in mammalian sperm by stimulatory agonists in the zona pellucida. Recently, it was shown that exocytosis is modulated in bovine sperm by extrinsic factors present in seminal fluids (Florman, H.M., and First, N.L. (1988) Dev. Biol. 128, 464-474). Fractionation of bovine seminal fluids yields a M(r) approximately 6,500, basic (pI approximately 8.5) peptide that accounts for the positive modulation of zona pellucida-induced acrosome reaction (ED50 and maximal response at 0.2 and 1 micrograms/ml, respectively). In addition, application of purified peptide to fura 2-loaded, cauda epididymal sperm supported zona pellucida-promoted elevations of Ca2+i equivalent to those observed with sperm treated with unfractionated seminal fluids in vitro or in vivo. Finally, peptide treatment regulated gamete interaction in a manner consistent with the distinct behaviors of cauda epididymal and ejaculated bovine sperm. This purified seminal peptide was identified by sequence analysis as caltrin, a previously characterized regulator of Ca2+ transport in bovine sperm. We therefore propose that caltrin is a regulator of sperm signal transduction pathways activated by zona pellucida binding.

Enzymatic dissection of the functions of the mouse egg's receptor for sperm.

During the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 (approximately equal to 80 kDa) serves as both receptor for sperm (J. D. Bleil and P. M. Wassarman, 1980c, Cell 20, 873-882) and inducer of the acrosome reaction (J. D. Bleil and P. M. Wassarman, 1983, Dev. Biol. 95, 317-324). In this investigation, small ZP3 glycopeptides (approximately equal to 1.5-6 kDa), obtained by extensive digestion of the purified glycoprotein with insoluble Pronase, were assayed for both sperm receptor and acrosome reaction-inducing activities. While ZP3 glycopeptides were virtually as effective as intact ZP3 in inhibiting binding of sperm to eggs in vitro ("receptor activity"), unlike intact ZP3, they failed to induce sperm to undergo the acrosome reaction. The latter was determined by indirect immunofluorescence using a monoclonal antibody directed against the acrosomal cap region of sperm. These results suggest that the sperm receptor activity of ZP3 is dependent only on its carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide chain of ZP3 as well.

Pharmacological properties of the T-type Ca2+ current of mouse spermatogenic cells.

The effects of pharmacological agents on the T-type Ca2+ current were studied in dissociated spermatogenic cells from the mouse. Ca2+ currents were elicited by depolarization in 10 mM Ca2+ and recorded in the whole-cell configuration of the patch clamp technique. The T-type current was inhibited by the following compounds: PN200-110 (IC50 = 4 x 10(-8) M) > nifedipine (IC50 = 4 x 10(-7) M) > pimozide (IC50 = 4.6 x 10(-7) M) > mibefradil (IC50 = 5 x 10(-6) M) > Ni2+ (IC50 = 3.4 x 10(-5) M) > verapamil (IC50 = 7 x 10(-5) M) > amiloride (IC50 = 2.4 x 10(-4) M) > Cd2+ (IC50 = 2.8 x 10(-4) M). However, the agents differed in the reversibility and the use dependence of their effects. Currents recovered rapidly and completely after removal of Ni2+, Cd2+, amiloride, or mibefradil, whereas recovery from verapamil block was rapid but incomplete. In contrast, we observed little recovery after the removal of pimozide and of the dihydropyridines (PN200-110, nifedipine). Moreover, mibefradil and pimozide exhibit a strongly use-dependent inhibition of current that is due to selective interaction of these drugs with the open state and the inactivated state of the channel, respectively, rather than with the resting state. These properties of the spermatogenic T-type Ca2+ channel differ from those of somatic cell T channels and suggest a molecular diversity of low voltage-activated Ca2+ channels.

Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis.

Previous work indicates that antagonists of the L-type voltage-dependent Ca2+ channel (VDCC) prevent the Ca(i) increase in mammalian sperm that is promoted by incubation in alkaline, K(+)-based media. Here, were provide additional evidence that sperm possess VDCC and show that their activation is required for the Ca2+ entry that mediates acrosomal exocytosis in both the presence and the absence of egg agonists. Specifically, we report that: (1) Sperm membrane potential changes, Ca(i) elevation, and acrosomal exocytosis have similar K+ dose dependencies, consistent with a characteristic requirement of a large depolarization for activation of the sperm VDCC; (2) High affinity binding sites (Kd approximately 0.35 +/- 0.03 and 0.45 +/- 0.06 nM; Bmax = 16.0 +/- 1.4 and 5.8 +/- 0.8 fmole/mg protein) for the VDCC antagonist, PN200-110, respectively, are present in membrane preparations from sperm of the ram and bull; (3) PN200-110 and the other VDCC antagonists nitrendipine, nisoldipine, verapamil, diltiazem, Ni2+, or Co2+ inhibit (IC50 = 0.1, 0.4, 0.6, 0.8, 1.0, 60, and 110 microM, respectively) the acrosomal exocytosis produced by combined elevation of pH0 and membrane depolarization; (4) Exocytosis induced by the ZP3 agonist of the mammalian egg also is inhibited by VDCC antagonists with similar dose dependencies; (5) Depolarizing treatments that presumably activate the sperm VDCC bypass the blockade of ZP3-induced exocytosis imposed by pertussis toxin. These results indicate that activation of the sperm VDCC is sufficient to induce sperm acrosomal exocytosis and that VDCC activation is necessary in the ZP3 signal transduction pathway. They also indicate that the presumed G-protein targets of pertussis toxin probably produce a required but indirect activation of the putative sperm VDCC. Possible intervening events include alteration of the voltage sensitivity of the VDCC, membrane depolarization, or both. We suggest that the depolarization-induced acrosome reaction may provide a useful system to investigate subsequent events in the exocytotic process.

Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida.

The sperm acrosome reaction is a Ca(2+)-dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies using ion-selective fluorescent probes suggested a role of voltage-sensitive Ca2+ channels in acrosome reactions. Here, wholecell patch clamp techniques are used to demonstrate the expression of functional T-type Ca2+ channels during mouse spermatogenesis. The germ cell T current is inhibited by antagonists of T-type channels (pimozide and amiloride) as well as by antagonists whose major site of action is the somatic cell L-type Ca2+ channel (1,4-dihydropyridines, arylalkylamines, benzothiazapines), as has also been reported for certain somatic cell T currents. In sperm, inhibition of T channels during gamete interaction inhibits zona pellucida-dependent Ca2+ elevations, as demonstrated by ion-selective fluorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca2+ channels to fertilization. In addition, the kinetics of channel inhibition by 1,4-dihydropyridines suggests a mechanism for the reported contraceptive effects of those compounds in human males.

A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs.

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at approximately 25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and approximately 20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.

An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis.

Solubilized oocyte zonae pellucidae promoted acrosomal exocytosis in fura-2- or carboxyfluorescein-loaded, mature bovine sperm. Associated elevations of internal [Ca2+] and pH in sperm suspensions were first detectable at 2-5 min, without apparent temporal resolution, and increased monotonically thereafter. Video imaging of fura-2-loaded, single cells identified a responsive subpopulation, destined to undergo exocytosis, that displayed no early transient but manifested lags of 1-7 min then sustained elevations of internal [Ca2+]. Both the zona-induced exocytosis and dye responses were diminished for functionally immature sperm and for mature sperm treated preliminarily with pertussis toxin. Together, these results indicate that a developmentally regulated mechanism of signal transduction employs G protein(s) to couple the physiological (zona) agonist to alterations of the internal ionic mediators of acrosomal exocytosis.