Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.     

Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm

Progesterone and 17 alpha-hydroxyprogesterone (but not other steroids such as testosterone, corticosterone, beta-estradiol, estrone, dehydroepiandrosterone, 20 alpha-hydroxypregnen-3-one, androstenedione, and pregnenolone) were shown to cause an immediate increase, in free cytosolic calcium ([Ca2+]i) in both capacitated and noncapacitated human sperm, using the fluorescent indicator fura 2. Significant increases in [Ca2+]i were observed with 10 ng/ml progesterone, while maximum effects were seen with 1 microgram/ml progesterone. Two other steroids 11 beta-hydroxyprogesterone and 5 alpha-pregnane-3,20-dione exhibited significant activity to increase [Ca2+]i. This increase in [Ca2+]i elicited by progesterone was entirely due to Ca2+ influx from the extracellular medium since the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA (2.5 mM) and the Ca2+ channel antagonist La3+ (0.25 mM) when added to the medium containing 2.5 mM Ca2+. Progesterone also stimulated the uptake of Mn2+ into sperm as measured by the quenching of fura 2 fluorescence. Progesterone has been found in human follicular fluid at levels capable of stimulating increases in [Ca2+]i. The similarities in responses induced by human follicular fluid and progesterone an increase in [Ca2+]i, and hence the acrosome reaction, is progesterone and/or 17 alpha-hydroxyprogesterone. Progesterone (1 microgram/ml) did not increase [Ca2+]i in somatic cells such as adipocytes, hepatocytes, Balb/c 3T3 cells, normal rat kidney, or DDT1 MF-2 cells. The effects of these progestins to increase [Ca2+]i, by activating a receptor-operated calcium channel, is the first report of such an activity in sperm. This phenomena possibly opens up a new field of steroid action in the area of sterility, fertility, and contraception at the level of the sperm.     

Increase of human spermatozoa intracellular Ca2+ concentration after fusion with prostasomes.

Prostasomes are membranous vesicles (150-200 nm diameter) present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and calcium, and some of their proteins are enzymes. Prostasomes are involved in a number of biological functions. In previous work, we discovered that prostasomes may fuse to sperm at neutral or at slightly acidic pH values. This mechanism may deliver calcium to sperm, thereby influencing sperm functions. We measured sperm [Ca2+]i with the fura-2 AM method and found that it increased after mixing prostasomes and sperm at pH values allowing fusion (pH 5-7). The increase of [Ca2+]i was proportional to the extent of fusion as measured through the relief of R18 self-quenching. We also examined the increase of sperm [Ca2+]i and the extent of fusion as a function of sperm to prostasome ratio and, also in this case, there was proportionality between the extent of fusion and the increase of [Ca2+]i that reached its maximal values in about 10-20 min. However, a detectable increase of [Ca2+]i was attained after 2 min of fusion. This would represent a new mechanism to influence sperm [Ca2+]i besides ion-exchange systems and ATP-dependent pumps. The value of [Ca2+]i remained elevated, unless Na+ was also present in the external medium. Therefore, the mechanism of fusion might influence deeply the physiology of sperm by producing a transient increase of [Ca2+]i.     

Studies related to progesterone-induced hamster sperm acrosome reaction

Experiments were designed to characterize the effect of progesterone on the hamster sperm acrosome reaction (AR). Progesterone stimulated exocytosis of previously capacitated spermatozoa in a dose-dependent manner Progesterone-3-(O-carboxymethyl)oxime:BSA conjugate also induced AR when added to capacitated sperm suspensions. EGTA and La³⁺, added 10 min before progesterone, completely abolished the steroid-stimulatory effect. Benzamidine, a trypsin inhibitor, also inhibited AR when added to sperm cells 10 min before progesterone. This effect was avoided when spermatozoa were treated with the Ca2+ ionophore ionomycin. Conversely, the H+ ionophore PCCP, or the Na⁺/K⁺ ionophore nigericin, did not prevent the effect of the inhibitor. Results suggest that progesterone acts on the hamster sperm plasma membrane to stimulate exocytosis, which requires external Ca2+ and presumably Ca2+ influx. In addition, a sperm trypsin like protease may be part of the mechanism by which progesterone stimulates AR. Since the ionomycin-induced AR does not require this proteolytic activity, the possible involvement of such an enzyme in the progesterone-stimulated Ca2+ influx necessary for the occurrence of AR is discussed. © 1996 Wiley-Liss, Inc.     

Alteration of intracellular [Ca2+] in sea urchin sperm by the egg peptide speract. Evidence that increased intracellular Ca2+ is coupled to Na+ entry and increased intracellular pH

The egg peptide speract increases intracellular pH (pHi) and cyclic nucleotides in sperm of the sea urchin Strongylocentrotus purpuratus by a mechanism dependent on seawater Na+ but not Ca2+ (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 2235-2241; Repaske, D. R., and Garbers, D. L. (1983) J. Biol. Chem. 258, 6025-6029). Using the Ca2+ indicators quin2 and indo-1, we show that speract stimulates a transient rise in intracellular [Ca2+] ([a2+]i) when millimolar Ca2+ is present in seawater. The rise is increased and extended by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), which also enhances 22Na+ uptake with or without Ca2+. Without MIX, speract initiates a rise in [Ca2+]i that peaks within approximately 5 s and decreases with a t1/2 of approximately 9 s. Activation of Na+:H+ exchange without speract by either Na+ addition to sperm in Na+-free seawater (NaFASW) or by monensin also increases [Ca2+]i, but neither change is transient. Inhibition of Na+:H+ exchange by increased seawater [K+] prevents the rise in [Ca2+]i initiated by either speract or Na+ addition to sperm in NaFASW. Increasing pHi by adding 10 mM NH4+ or by addition of Li+ to sperm in NaFASW does not increase [Ca2+]i. The data suggest that speract binding leads to rapid activation of Na+:H+ exchange; and, as a consequence, [Ca2+] entry increases transiently through either Na+:Ca2+ exchange or else through a verapamil-insensitive Ca2+ channel. MIX prevents the inactivation of this entry mechanism.     

Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.     

Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2

The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, [Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the [Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on [Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.     

Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm

Cell surface receptors for progesterone were visualized in human sperm using fluorescein isothiocyanate-progesterone 3-(O-carboxymethyl) oxime-bovine serum albumin (FITC prog CMO BSA). The receptors were confined to the head and not the midpiece or tail. FITC prog CMO BSA was also an effective stimulus to elevate intracellular free calcium in human sperm as detected by fura-2 fluorescence. The elevation of intracellular free calcium is a stimulus for the acrosome reaction, a process which is necessary to occur for sperm to fertilize the egg. It is proposed that progesterone, which is present in the female reproductive tract, can bind to progesterone receptors located in the plasma membrane of the sperm head and elicit an influx of Ca2+ into the underlying cytoplasm and or acrosome and induce the acrosome reaction and facilitate fertilization.     

Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms

Previous work (Babcock, D. F., Rufo, G. A., and Lardy, H.A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1327-1331) established that increased cytosolic pH (pHi) promotes metabolic and swimming activity of bull sperm and that intracellular alkalinization results from elevated extracellular K+, presumably as a consequence of membrane depolarization. The present studies show that a persistent but reversible increase in [Ca2+]i accompanies the increase in pHi that similarly results from treatment of ram sperm with elevated [K+] in alkaline media. Because comparable increases in pHi occur in the presence or absence of external Ca2+ and because [Ca2+]i is unaltered by imposed changes in pHi alone, [Ca2+]i and pHi apparently are neither directly linked by transmembrane Ca2+/H+ exchange nor indirectly linked through Na+/H+ and Na+/Ca2+ exchange under these conditions. Instead, inhibition of K+-induced increases in [Ca2+]i (but not of increases in pHi) by prenylamine, diltiazem, nifedipine, or verapamil (C1/2 = 6, 20, 30, and 60 microM, respectively) indicates that voltage-dependent Ca2+ channels, distinct from previously described voltage-dependent effectors of pHi, operate in mammalian sperm to control [Ca2+]i. Treatment with Cs+ plus valinomycin (as an alternative method of membrane depolarization) increases pHi much more effectively than it increases [Ca2+]i, and thus also partially supports this contention. In contrast to an apparent insensitivity to pHi, K+-dependent increases in [Ca2+]i are promoted reversibly by elevation of pHo, probably reflecting local surface charge effects on channel activity (as suggested by patch-clamp studies in other systems). A selective increase in membrane permeability to Ca2+ that is induced by 12 mM NaF under nondepolarizing conditions is not a consequence of cellular aggregation, but is attenuated by the chelator deferoxamine, suggesting that GTP-binding protein additionally may couple sperm Ca2+ channels to surface receptors and promote channel opening during sperm capacitation, presumably in response to agonists produced within the mammalian female reproductive tract.     

Non-genomic immunosuppressive actions of progesterone inhibits PHA-induced alkalinization and activation in T cells

Progesterone is an endogenous immunomodulator, and can suppress T-cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Therefore, the aim of this study was to investigate whether the non-genomic effects on ion fluxes by progesterone could immunosuppress phytohemagglutinin (PHA)-induced human peripheral T-cell activation. The new findings indicated that, first, only progesterone stimulated both [Ca2+]i elevation and pHi decrease; in contrast, estradiol or testosterone stimulated [Ca2+]i elevation and hydrocortisone or dexamethasone stimulated pHi decrease. Secondly, the [Ca2+]i increase by progesterone was dependent on Ca2+ influx, and the acidification was blocked by Na+/H+ exchange (NHE) inhibitor, 3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE-694) but not by 5-(N,N-dimethyl)-amiloride (DMA). Thirdly, progesterone blocked phorbol 12-myristate 13-acetate (PMA) or PHA-induced alkalinization, but PHA did not prevent progesterone-induced acidification. Fourthly, progesterone did not induce T-cell proliferation; however, co-stimulation progesterone with PHA was able to suppress PHA-induced IL-2 or IL-4 secretion and proliferation. When progesterone was applied 72 h after PHA stimulation, progesterone could suppress PHA-induced T-cell proliferation. Finally, immobilization of progesterone by conjugation to a large carrier molecule (BSA) also stimulated a rapid [Ca2+]i elevation, pHi decrease, and suppressed PHA-induced proliferation. These results suggested that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress the genomic responses to PHA. Progesterone might act directly through membrane specific nonclassical steroid receptors to cause immunomodulation and suppression of T-cell activation during pregnancy.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10-30 min of ischemia followed by 5-30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(-)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon beta-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.     

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O2-) release, membrane depolarization and an increase in cytoplasmic free Ca2+, [Ca2+]i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca2+ ionophore ionomycin was used as stimulus, an increase in [Ca2+]i clearly preceded the onset of membrane depolarization, which was followed by O2- release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutinin was used as stimulus, no demonstrable lag was seen in any of the responses. O2- release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells with a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca2+]i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O2- release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca2+]i was not affected. The dose-response curves for triggering of O2- release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca2+]i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O2- release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca2+]i, but not by augmenting an increase in [Ca2+]i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.     

Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida

The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]i increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+]i occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]i was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle approximately 20 s after the onset of the [Ca2+]i rise. High-resolution imaging revealed that the [Ca2+]i rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]i rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+]i rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR.     

Ca2+ oscillations induced by hormonal stimulation of individual fura-2-loaded hepatocytes

Isolated rat hepatocytes were loaded with the Ca2+ indicator fura-2 to measure cytosolic free Ca2+ concentrations ([Ca2+]i) in individual cells by digital ratio imaging microscopy. Stimulation with 0.1 nM vasopressin, 0.5 microM phenylephrine, or 0.5 microM ATP caused repetitive spikes of high [Ca2+]i in a high percentage of cells, in agreement with Woods et al. (Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 319, 600-602), but unlike the results of Monck et al. (Monck, J. R., Reynolds, E. E., Thomas, A. P., and Williamson, J. R. (1988) J. Biol. Chem. 263, 4569-4575). Reduction in extracellular [Ca2+] decreased the frequency but not the amplitude of the spikes, suggesting that the spikes result from dumping of intracellular stores and that the entry of extracellular Ca2+ affects only the rate of replenishment of those stores. Membrane depolarization failed to elevate [Ca2+]i and had an effect similar to removal of extracellular Ca2+ in decreasing the frequency of agonist-evoked [Ca2+]i oscillations or inhibiting them altogether, arguing against any significant role for voltage-operated Ca2+ channels.     

Intracellular free Ca2+ changes during physiological polyspermy in amphibian eggs.

We have made the first measurements of intracellular free calcium activity ([Ca2+]i) in urodele eggs during physiological polyspermic fertilization. Jellied eggs of the urodele amphibian Pleurodeles waltlii were impaled with intracellular Ca(2+)-selective microelectrodes and inseminated under various conditions of sperm:egg ratio to obtain various degrees of polyspermy. In 17 out of 45 cases the egg [Ca2+]i level (0.41 microM) showed no variation following fertilization. In 28 other cases, however, the egg displayed a slow increase in [Ca2+]i of 0.15 microM, starting around 15 minutes after fertilization and reaching a plateau level around 10 minutes later. The amplitude of the fertilization-associated increase in [Ca2+]i was found to be independent of the number of sperm interacting with the egg surface. Measurements with two Ca(2+)-microelectrodes impaled in single eggs showed that the increase in [Ca2+]i did not simultaneously occur at distinct places within the egg cortex and, in some cases, could be detected by only one of the two microelectrodes. This latter observation, as well as the absence of [Ca2+]i change at fertilization in some experiments, strongly suggested that each sperm interacting with the egg might, at various places, trigger a localized, non-propagating change in [Ca2+]i. Experiments in which eggs were locally inseminated, using a micropipette directed towards the site of impalement of one of the two Ca(2+)-microelectrodes, clearly established that [Ca2+]i changes, although incapable of propagating over the entire egg cortex, might nevertheless travel very slowly over short distances, their amplitude vanishing rapidly as they propagate from around the sites of sperm entry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular ATP itself elicits superoxide generation in guinea pig peritoneal macrophages.

Extracellular ATP itself elicited the generation of superoxide (O2-) in guinea pig peritoneal macrophages associated with an increase in cytosolic calcium ([Ca2+]i). The ATP-induced O2- generation was completely inhibited by pretreatment with pertussis toxin (PT) accompanied by the suppression of [Ca2+]i mobilization. Pre-exposure to a small amount of phorbol myristate acetate (PMA) primed the ATP-induced generation of O2- without a change of [Ca2+]i. The results suggest that ATP-induced O2- generation is mediated by [Ca2+]i mobilization and by PT-sensitive G protein.     

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.     

Spatiotemporal analysis of [Ca2+]i rises in mouse eggs after intracytoplasmic sperm injection (ICSI)

Intracytoplasmic sperm injection (ICSI) into mammalian eggs induces repetitive rises in intracellular Ca2+ concentration ([Ca2+]i) which are the pivotal signal in fertilization. Spatiotemporal aspects of [Ca2+]i rises following ICSI into the periphery of mouse eggs were investigated with high-speed confocal microscopy. The first Ca2+ response was generated 25-30 min after ICSI, when [Ca2+]i increased slowly and reached a certain level. The [Ca2+]i rise occurred synchronously over the ooplasm, attained the peak in 40-70 s, and lasted for 5-7 min. Succeeding Ca2+ responses occurred at intervals of 20-30 min, associated with the faster rate of [Ca2+]i rise and the shorter duration as Ca2+ oscillations progressed. The [Ca2+]i rises took the form of a wave that started from an arbitrary cortical region, but not from the vicinity of the injected sperm head. The Ca2+ wave became more pronounced and propagated across the egg faster in the later Ca2+ responses. An artifactual [Ca2+]i rise was inevitably produced during the ICSI procedure. The larger artifact affected the subsequent first Ca2+ response, resulting in the faster [Ca2+]i rise (time to peak, 10-20 s), slight spatial heterogeneity of [Ca2+]i rise in the ooplasm (but not a wave) and the shorter duration (3-4 min). The artifact slightly affected the amplitude of the second Ca2+ response, but little affected the later Ca2+ responses. It is suggested that the factor(s) that leaked out of the injected spermatozoon diffuses to a wide area and sensitizes Ca2+ channels of the endoplasmic reticulum to induce Ca2+ release synchronously over the ooplasm. The enhanced sensitization leads to propagating Ca2+ release initiated from the cortex that is more sensitive to the sperm factor.     

Speract induces calcium oscillations in the sperm tail

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.