Progesterone-induced increase of sperm cytosolic calcium is enhanced by previous fusion of spermatozoa to prostasomes

Ejaculated spermatozoa must undergo a number of modifications before becoming able to fertilize the oocyte. The interaction of sperm with other semen components may influence these phenomena; human semen contains vesicles of prostatic origin, called prostasomes that may fuse to sperm at slightly acidic to neutral pH values.Prostasomes contain calcium and it has been demonstrated that their fusion with spermatozoa produces a transient increase (wave) of [Ca(2+)](i) in these cells. The fusion process also transfers protein and lipid to spermatozoa. These phenomena may induce long-lasting changes of sperm properties. We test the hypothesis that spermatozoa, as modified by fusion, change their ability to undergo the progesterone-induced increase of [Ca(2+)](i) and we find that the increase of [Ca(2+)](i) produced by the fusion with prostasomes and by the stimulation with progesterone are independent and additive phenomena. We also find that spermatozoa present a stronger response to the progesterone-induced increase of [Ca(2+)](i) if they are previously made to fuse with prostasomes. This effect does not depend directly on the [Ca(2+)](i) increase due to fusion, since it is still present after the [Ca(2+)](i) has returned to resting values.     

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.     

The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.     

Rapid inhibition of Ca2+ influx by neurosteroids in murine embryonic sensory neurones

The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ + Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50 mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

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.     

Cl-]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.     

Single pancreatic beta-cells from normal rats exhibit an initial decrease and subsequent increase in cytosolic free Ca2+ in response to glucose.

Since it was reported that glucose stimulation initially lowers as well as subsequently raises the cytosolic free calcium concentration [( Ca2+]i) in pancreatic islet cells from hyperglycemic ob/ob mice, it has been argued whether the lowering of [Ca2+]i is physiological or artifactual. In the present study, [Ca2+]i in single pancreatic beta-cells from normal rats was measured by Fura-2 microfluorometry. Following elevation of the glucose concentration from 2.8 mM (basal) to 16.7 mM, a bimodal change in [Ca2+]i, an initial decrease and subsequent increase, was demonstrated. When the basal glucose concentration was raised to 5.6 mM, the stimulation with 16.7 mM glucose also induced the decrease in [Ca2+]i in the majority of the cells, though the amplitude of the decrease was reduced. An elevation of the glucose concentration from 2.8 to 5.6 mM induced the decrease in [Ca2+]i but not usually the increase in [Ca2+]i. Removal of extracellular Ca2+ eliminated the increase in [Ca2+]i without affecting the decrease in [Ca2+]i. Thus, the decrease and increase in [Ca2+]i were clearly dissociated under certain conditions. In contrast, mannoheptulose (an inhibitor of glucose metabolism) inhibited both the decrease and increase in [Ca2+]i. These results demonstrate that the glucose-induced bimodal change in [Ca2+]i is a physiological response of islet beta-cells, and that the decrease and increase in [Ca2+]i are generated by mutually-independent mechanisms which are operated through glucose metabolism by islet beta-cells.     

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.     

The development of signal transduction pathways during epididymal maturation is calcium dependent

Capacitation has been correlated with the activation of a cAMP-PKA-dependent signaling pathway leading to protein tyrosine phosphorylation. The ability to exhibit this response to cAMP matures during epididymal maturation in concert with the ability of the spermatozoa to capacitate. In this study, we have addressed the mechanisms by which spermatozoa gain the potential to activate this signaling pathway during epididymal maturation. In a modified Tyrode's medium containing 1.7 mM calcium, caput spermatozoa had significantly higher [Ca2+]i than caudal cells and could not tyrosine phosphorylate in response to cAMP. However, in calcium-depleted medium both caput and caudal cells could exhibit a cAMP-dependent phosphorylation response. The inhibitory effect of calcium on tyrosine phosphorylation was also observed in caudal spermatozoa using thapsigargin, a Ca(2+)-ATPase inhibitor that increased [Ca2+]i and precipitated a corresponding decrease in phosphotyrosine expression. We also demonstrate that despite the activation of tyrosine phosphorylation in caput spermatozoa, these cells remain nonfunctional in terms of motility, sperm-egg recognition and acrosomal exocytosis. These results demonstrate that the signaling pathway leading to tyrosine phosphorylation in mouse spermatozoa is negatively regulated by [Ca2+]i, and that maturation mechanisms that control [Ca2+]i within the spermatozoon are critically important during epididymal transit.     

Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents

The objectives of the present study were to clarify and compare the characteristics of the transient rises in intracellular calcium concentrations ([Ca2+]i) induced either by spermatozoa or by stimulation with artificial activators in bovine oocytes. These transient rises in [Ca2+]i in oocytes matured in vitro were recorded with Ca2+ imaging using the Ca2+ indicator fura-2. During fertilization, a series of transient rises in [Ca2+]i was observed. The first Ca2+ response peaked at a concentration of 521 +/- 39 nM (n = 20) and lasted for 4 min, while the subsequent Ca2+ responses were significantly smaller and shorter, with a peak of 368 +/- 13 nM (n = 23) and a duration of 2 min. Injection of inositol 1,4,5- triphosphate (InsP3) into unfertilized oocytes caused a transient rise in [Ca2+]i in a dose-dependent manner. The maximum response was induced by 20 nA x 1 sec injection of InsP3. Thimerosal, a sulfhydryl reagent, induced the repetitive transient rises in [Ca2+]i. The peak and the duration of the rises in [Ca2+]i induced by InsP3 or thimerosal were smaller and shorter, respectively, than those of the first rise induced by spermatozoa. Ethanol and Ca2+ ionophore IA23187, which are general parthenogenetic activators of unfertilized oocytes, each induced a single transient rise in [Ca2+]i. The duration of the rise in [Ca2+]i by ethanol or Ca2+ ionophore was significantly longer than that by spermatozoa at fertilization, although the peaks were smaller. These results clarified the characteristics of the rises in [Ca2+]i induced by spermatozoa and by several artificial reagents, and showed that the first rise in [Ca2+]i induced by spermatozoa had a higher peak [Ca2+]i and a longer duration compared with each the subsequent rises in [Ca2+]i and the rises in [Ca2+]i induced by artificial reagents. These indicate that a mode like as the first rise in [Ca2+]i induced by spermatozoa is an effective trigger for artificial activation of oocytes.     

Desynchronising effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries

The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs.     

What Is the Core Oscillator in the Speract-Activated Pathway of the Strongylocentrotus purpuratus Sperm Flagellum?

Sperm chemotaxis has an important role in fertilization. Most of our knowledge regarding this phenomenon comes from studies in organisms whose fertilization occurs externally, like sea urchins. Sea urchin spermatozoa respond to sperm-activating peptides, which diffuse from the egg jelly coat and interact with their receptor in the flagellum, triggering several physiological responses: changes in membrane potential, intracellular pH, cyclic nucleotide levels, and intracellular Ca2+ concentration ([Ca2+]). In particular, flagellar [Ca2+] has been shown to oscillate. These [Ca2+] oscillations are correlated with changes in the flagellar shape and so with the regulation of the sperm swimming paths. In this study, we demonstrate, from a mathematical modeling perspective, that the reported speract-activated signaling pathway in Strongylocentrotus purpuratus (speract being a sperm-activating peptide specific to this species) has the necessary elements to replicate the reported [Ca2+] oscillations. We further investigate which elements of this signaling pathway constitute the core oscillator.     

Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose

Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calcium (1 mM) was added at T = 15 min, and heparin (HEP; 10 micrograms/ml) or heparin plus glucose (hGLUC; 5 mM in 10 micrograms/ml heparin) was added at T = 30 min. Spermatozoal Ca2+ content and regulation differed among males (P = 0.0066). Relative initial [Ca2+]i differed significantly among all stages of maturity (0.84 +/- 0.104, 0.76 +/- 0.023, 1.20 +/- 0.036 LSM of relative Ca2+ units for caput, cauda and ejaculate spermatozoa respectively; P = 0.001). Rate of Ca2+ uptake was similar for ejaculate and cauda spermatozoa (0.021 +/- 0.005 and 0.026 +/- 0.002 relative Ca2+ units/sec) but slower for caput spermatozoa (0.012 +/- 0.001; P = 0.0006). There was no immediate effect of HEP or hGLUC in any stage (P > 0.05), and caput spermatozoa did not differ from cauda spermatozoa for any treatment or time period. A significant increase in [Ca2+]i was seen in ejaculate spermatozoa treated with HEP from 2 h on (P < 0.05). This study demonstrates that both the absolute Ca2+ concentration and the rate of Ca2+ internalization in equine spermatozoa is dependent on the stage of maturation. Ejaculate spermatozoa respond to heparin through increased [Ca2+]i, which may play a role in the fertilizing ability of ejaculate spermatozoa.     

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.     

Cell surface-binding sites for progesterone mediate calcium uptake in human sperm.

Recent studies (e.g. Blackmore, P. F., Beebe, S. J., Danforth, D. R., and Alexander, N.) (1990) J. Biol. Chem. 265, 1376-1380) have shown that in human sperm, progesterone produces a rapid increase in intracellular free calcium ([Ca2+]i) and an induction of the acrosome reaction (e.g. Osman, R. A., Andria, M. L., Jones, A. D., and Meizel, S. (1989) Biochem, Biophys. Res. Commun. 160, 828-833). In this study, the location of progesterone receptors on the cell surface of human sperm was identified using progesterone immobilized on bovine serum albumin (BSA) (progesterone 3-(O-carboxymethyl)oxime:BSA) as well as progesterone and its 3-O-carboxymethyloxime derivative. Using fluorescence microscopy, BSA-fluorescein isothiocyanate was shown to be excluded from intact sperm, thus validating the use of progesterone 3-(O-carboxymethyl)oxime:BSA to identify cell surface-binding sites for progesterone. The immobilized progesterone and the 3-O-carboxymethyloxime derivative rapidly increased [Ca2+]i and were full agonists, although they were approximately 1.5 orders of magnitude less potent than progesterone. They also displayed an identical time course to increase [Ca2+]i as free progesterone, and the entire increase in [Ca2+]i was due to the influx of Ca2+. This progesterone-mediated response displayed different steroid receptor characteristics since the very potent inhibitors of genomic progesterone responses, RU38486 and ZK98.299, were very ineffective at inhibiting the progesterone-mediated increase in [Ca2+]i. Also the synthetic progestins megestrol, medroxyprogesterone acetate, norgestrel, norethynodrel, norethindrone, R5020, and cyproterone acetate did not mimic the effects of progesterone to increase [Ca2+]i. It is proposed that a distinct nongenomic cell surface receptor for progesterone exists in human sperm.     

Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca(2+) by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca(2+)](i). The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca(2+)](i). Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca(2+) in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca(2+)](i), resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca(2+) in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.     

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.