Calcium-free Medium Research Articles

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Androgens Rapidly Activate Nuclear Factor-Kappa B via Intracellular Ca2+ Signalling in Human Vascular Endothelial Cells

There exists a striking gender difference in the incidence of atherosclerosis. Androgen exposure may predispose men to earlier onset atherosclerosis. We previously demonstrated that the potent androgen, dihydrotestosterone, enhanced the binding of monocytes to endothelial cells, via androgen receptor/nuclear factor kappa B-dependent expression of the cell adhesion molecules, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1. We now show that testosterone and dihydrotestosterone can also induce a novel, non-genomic pathway that leads to the rapid activation of nuclear factor-kappa B via intracellular Ca2+ signalling, initiated at the plasma membrane. Human umbilical vein endothelial cells exposed to 6-60 nM testosterone or dihydrotestosterone showed a rapid increase in intracellular calcium levels. The testosterone or dihydrotestosterone effect on increased intracellular calcium could not be abrogated by pre-incubation with androgen receptor antagonist, hydroxyflutamide, or by U73122, an inhibitor of intracellular calcium release from endoplasmic reticulum stores. However, pre-incubation with both Ni2+or an extracellular Ca2+ chelator blocked the testosterone-induced intracellular Ca2+ surge. Testosterone conjugated to bovine serum albumin was equal to free testosterone in its ability to induce the intracellular Ca2+ surge. Binding studies showed testosterone does bind to the plasma membrane, however, classical androgen receptor was unable to be detected in the plasma membrane of human umbilical vein endothelial cells. Testosterone was found to rapidly increase nuclear factor-kappa B activity, an effect that was blocked when cells were incubated in calcium-free media. This study demonstrates for the first time that testosterone induces a non-genomic membrane-initiated Ca2+ dependent signalling pathway that leads to the rapid activation of nuclear factor-kappa B.

184 INHIBITION OF CALCIUM EFFLUX EXTENDS THE DURATION OF CALCIUM SIGNALS IN PIG OOCYTES

Calcium signaling involves the transient elevation in the intracellular free-calcium concentration, which is responsible for controlling a great number of biological functions. In many cell types, a signal is generated when calcium stored in the endoplasmic reticulum is released into the cytoplasm, followed by an influx of calcium across the plasma membrane. At the same time, calcium is removed from the cytosol by ATPases, which pump it back into the intracellular store or out of the cell. The size of the calcium signal is thus determined by the amount of calcium moving into and out of the cytoplasm. In the present study, we investigated the effect of inhibiting the movement of calcium across the plasma membrane on a calcium-signal that was artificially induced in pig oocytes. In vitro-matured pig oocytes were loaded with the calcium-indicator dye, fura-2. The release of calcium from the cytoplasmic stores was stimulated by adding ethanol at a final concentration of 7% and changes in the intracellular free-calcium levels were monitored by using InCyt Im2, a dual-wavelength fluorescence imaging system. In the control group, fluorescent measurements were performed in the presence of extracellular calcium. In additional treatment groups, the ethanol treatment was performed in (1) a calcium-free medium (prevents calcium influx); (2) the presence of 1 mM gadolinium (limits calcium fluxes across the plasma membrane in both directions); and (3) the presence of gadolinium in a calcium-free medium. In each group, 15 oocytes were measured; the data were subjected to 1-way ANOVA and differences between treatment means were compared by the Tukey's test. We found that in control oocytes, 7% ethanol induced a rise of 722.1 ± 49.2 nM in the cytoplasmic calcium levels and the average duration of the calcium transient was 309.1 ± 11.3 s. In the calcium-free medium, this increase was significantly smaller (only 117.9 ± 4.8 nM; P < 0.01), probably because the calcium release was not followed by an influx of calcium across the plasma membrane. In the presence of 1 mM gadolinium in the regular calcium-containing medium, the intracellular calcium levels climbed by 278.8 ± 45.4 nM and dropped to baseline levels only after 773.3 ± 79.9 s. Finally, in the calcium-free medium and in the presence of 1 mM gadolinium, 7% ethanol induced only a small rise (64.5 ± 6.3 nM) in the cytoplasmic calcium levels. These latter increases were both significantly lower (P < 0.01) compared to that in the control group. The results indicate that (1) intracellular calcium signals are sustained by a calcium influx from the extracellular medium and (2) preventing calcium efflux extends the duration of the cytoplasmic calcium elevation in the oocyte. This may have relevance in the manipulation of intracellular calcium levels in oocytes for the development of novel parthenogenetic activation methods.

Change in Ca2+-responses of cultivated brown adipocytes at adrenergic activation

The thermogenic capability of brown adipose tissue is controlled by noradrenaline. By interacting with α1- and β-adrenoreceptors of adipocytes, noradrenaline (NA) increases the intracellular concentration of Ca2+ ([Ca2+]i) and cAMP. The changes in [Ca2+]i under the action of NA and selective agonists of α1- and β-adrenoreceptors, i.e., cirazoline and isoproterenol (IP), are recorded on individual cells of the primary culture of adipocytes during the day in vitro (DIV) 1, DIV 3, and DIV 6. The change in [Ca2+]i under the effect of IP as compared to the response to cirazoline in cells of DIV 1 is characterized by a higher amplitude and shorter duration of impulses in the entire diapason of the used physiological concentrations. After DIV 3, these differences are insignificant and, after DIV 6, the differences in kinetics are nearly absent. For all three agonists, the kinetics of the [Ca2+]i change in the proliferating and differentiated cells is significantly different; i.e., the response amplitude increases with the age of the culture and the duration of transitory response decreases, while sensitivity to agonists of adrenoreceptors increases. It can be seen from the rise in [Ca2+]i with an inhibitor of Ca2+-ATPase of the endoplasmic reticulum thapsigargin in calcium-free medium that the source of calcium ions in the endoplasmic reticulum rises with the growth and development of cells in culture, while the rate at which Ca2+ is pumped out of cells, which characterizes the activity of Ca2+-ATPase of the plasma membrane, increases.

Histamine Elevates Free Intracellular Calcium in Mouse Retinal Dopaminergic Cells via H1-Receptors

Previously, retinopetal axons containing histamine and dopaminergic neurons expressing histamine H(1)-receptor had been localized in mouse retinas using anatomic techniques. The goal of these experiments was to demonstrate that these receptors are functional. Dopaminergic cells were acutely isolated from retinas of transgenic mice expressing red fluorescent protein under control of the tyrosine hydroxylase promoter and loaded with the calcium indicator Fura-2. Under control conditions, there were spontaneous oscillations in the levels of free intracellular calcium in dopaminergic cells. These oscillations were abolished in nominally calcium-free extracellular medium and in 1 μM tetrodotoxin, findings suggesting that the oscillations were mediated by calcium entry across the plasma membrane in response to sodium-dependent action potentials. Histamine increased the mean free intracellular calcium in the dopaminergic cells by increasing the frequency and/or amplitude of the calcium oscillations. The effects of histamine were dose-dependent and reached maximum at 5 μM. With this dose, there was a 65% increase in the mean free intracellular calcium concentration. The histamine H(1)-receptor antagonist, pyrilamine, blocked the effects of 5 μM histamine when applied at 50 μM. The selective histamine H(1)-receptor agonists, 2-(3-trifluoromethylphenyl) histamine and methylhistaprodifen significantly increased mean free intracellular calcium when applied at 5 μM. Histamine released from retinopetal axons in the mouse retina can elevate intracellular calcium levels in the perikarya of dopaminergic cells via the activation of histamine H(1)-receptors.

Modulatory role of magnesium on the contractile response of rat aorta to several agonists in normal and calcium-free medium.

Acute withdrawal of external Mg2+ increased basal tone of rat isolated aorta incubated in the presence of Ca2+. Above normal levels of Mg2+ (1-4 mM) inhibited basal tone while much higher levels of the divalent cation (64-256 nM) evoked contractile responses regardless of the presence of Ca2+. Contractile responses to noradrenaline (1 microM) and KCl (80 mM) were inhibited by addition of cumulative concentrations of Mg2+. Acetylcholine-induced contractions in the presence of physiological concentrations of Mg2+ (1 mM) decreased gradually to the basal tone, but a sustained contraction was observed in the absence of this ion. In Ca(2+)-free medium, acetylcholine-induced phasic responses indicate the existence of an acetylcholine-sensitive Ca2+ store. KCl induced contraction only in Krebs solution, although a small residual contraction could be observed in Ca(2+)-free medium in some experiments. Mg(2+)-depletion in the extracellular medium increased contractile responses induced by acetylcholine and KCl in Ca(2+)-free medium. These results suggest that extracellular Mg2+ modulates basal tone, Ca2+ channels and responsiveness to various agents in the absence of Ca2+.

Comparison of the effects of hydralazine and nifedipine on contractions and 45Ca influx of rat aorta

The effect of the vasodilator hydralazine has been compared with nifedipine on KCl-(K+) (60 mM) and noradrenaline-(NA) (10 microM) induced 45Ca uptake and contractile responses in rat aorta arterial strips without endothelium. Hydralazine (0.5-10 mM) was equally effective in relaxing K(+)- (IC50 = 2.2 +/- 0.17 mM) and NA- (IC50 = 3.06 +/- 0.25 mM) induced tension, the degree of relaxation depending on the dose. Nifedipine totally inhibited K(+)- (IC50 = 3.16 +/- 0.28 nM) induced contractions with lower doses than were necessary to relax (up to 54.0 +/- 4.1% with supramaximal concentrations) NA-induced contractions (IC50 = 1.48 +/- 0.12 microM). In the experiments in a calcium-free medium, nifedipine (1 microM) had no effect on the NA- (10 microM) induced contractions whereas hydralazine (1 mM) strongly inhibited them. Nifedipine did not affect the basal uptake of 45Ca but the induced uptakes were reduced to 66.3 +/- 3.2% (K+) and 65.5 +/- 4.1% (NA) of their basal values. Hydralazine did not affect the basal uptake of 45Ca nor that induced by the two vascoconstrictor agents. These results suggest that nifedipine acts on the cell membrane by blocking the movements of calcium through the voltage-dependent and receptor-operated calcium channels, whilst hydralazine has an intracellular effect.

Spasmolytic action of adenosine on the guinea-pig ileum.

Abstract Adenosine (10−5M) reduces the contractile response of the guinea-pig isolated ileum to 5-hydroxytryptamine or barium more than the responses to acetylcholine or histamine. Adenosine also inhibits the contractile response of the guinea-pig ileum to a calcium-free medium. These results suggest that adenosine effects a blockade of the indirect responses to barium or 5-hydroxytryptamine mediated through intrinsic nerves and has a weak direct inhibitory effect on the smooth muscle cells of this tissue.

Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.

Inorganic phosphate stimulates apoptosis in murine MO6-G3 odontoblast-like cells

ObjectiveDental pathologies such as caries are the most prevalent disease worldwide with infectious and social complications. During the process of caries formation, the tooth is degraded and demineralization of enamel and dentine leads to the release of large amounts of inorganic phosphate (Pi) within dental tubuli. As Pi has been shown to induce apoptosis in skeletal cells, including osteoblasts and chondrocytes, we questioned whether high concentrations of Pi could affect odontoblast viability, proliferation and apoptosis. DesignUsing the odontoblast-like MO6-G3 cell line as a model, we used cell counting and MTS-based colorimetric assays to measure cell viability and proliferation. Apoptosis was assessed using Hoechst nuclei staining and detection of the early apoptotic markers annexin V and Apo2.7. ResultsWe show for the first time that a high Pi concentration (7mM) induced a decrease in odontoblast viability and proliferation together with a large increase in apoptosis. These effects were blunted in calcium-free medium, possibly due to the formation of calcium-phosphate crystals in the presence of high Pi concentrations. ConclusionThis study contributes to clarifying the effect of Pi on odontoblast viability and apoptosis, which may improve our understanding of the role of Pi during caries formation.

Involvement of cAMP in the Spasmolytic Effect of Diclofenac on Rat Uterine Smooth Muscle

Diclofenac produced dose-dependent relaxation on vanadate-induced contraction of rat uterus incubated in calcium-free medium plus EDTA. This effect was antagonized by the inhibitors of cAMP-dependent protein kinases Rp-adenosine 3′,5′-monophosphothionate, triethylamine salt (1, 10 and 100 μM) and N-p-tosyl-L-phenylalanine-chloromethyl-ketone (1 and 10 μM). The inhibitor of phosphatases (okadaic acid, 0·1 and 0·3 μM) also reduced the effect of diclofenac. Our results suggest that the spasmolytic effect of diclofenac is mediated by cAMP.

Non-quantal release of acetylcholine in guinea-pig airways: role of choline transporter

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.

Regulation of Ca2+ influx in proliferating and differentiating murine myoblasts with participation of L-type Ca2+ channels

The basic mechanisms of regulation of Ca2+ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca2+ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca2+ through these channels is regulated by the adrenergic system. The influx of Ca2+ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca2+ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca2+ influx is substantially lower than in proliferating cells, and maximal influx of Ca2+ may be reached only upon exhaustion of reticular stocks.

Nad(P)H Oxidase Regulate Glut4 Traffic by Intracellular Calcium Release in Insulin Stimulated L6-Glut4myc Skeletal Muscle Cell

Insulin-regulated GLUT4 traffic have been widely explored, but the role of calcium release and ROS signaling in this process is poorly understood. We studied insulin-induced GLUT4 traffic in stable expressing GLUT4myc skeletal muscle cells using singe cell imaging and quantification of extracellular myc exposure. Exofacial exposure of myc epitope, glucose uptake and AktS473 activation induced by insulin were independent of extracellular calcium. Pre-incubation with the intracellular calcium chelator BAPTA inhibited activation of Akt, p70S6K and ERK1 but not ERK2. On the other hand, p-ERK1/2 was inhibited by 2-APB, an IP3R inhibitor. The intracellular calcium chelators BAPTA and NES-PV-DsRed (cytosol directed parvalbumin) respectively reduced insulin-dependent extracellular exposure of myc. Moreover, 50µM ryanodine and 20 µM 2-APB partially inhibited the exofacial exposure of myc with an additive effect. In myotubes normal media, the calcium ionophore ionomycin, induced an increase in extracellular exposure of myc and was additive to insulin. In contrast, in myotubes maintained in calcium-free media, the effect of insulin was totally inhibited by ionomycin. Insulin increased H2O2 production measured with the cytosolic molecular sensor HyPer and this effect was inhibited by NAC pre-treatment. p47 subunit of NADP(H)ox labeled differentiated myotubes with a striated pattern but was absent in myoblasts. Both antioxidant agents, NAC and trolox partly reduced the externalization of myc as did the NADP(H)ox inhibitor apocynin. These data suggest that insulin induces an increase in NADP(H)ox activity and activation by ROS of RyR and IP3R intracellular calcium channels to foster GLUT4 translocation.FONDAP 15010006, FONDECYT 3110170.

Effect of Efonidipine on TGF-β1–Induced Cardiac Fibrosis Through Smad2-Dependent Pathway in Rat Cardiac Fibroblasts

Transforming growth factor beta-1 (TGF-β1) plays a critical role in progression of cardiac fibrosis, which may involve intracellular calcium change. We examined effects of efonidipine, a dual T-type and L-type calcium channel blocker (CCB), on TGF-β1-induced fibrotic changes in neonatal rat cardiac fibroblast. T-type and L-type calcium channel mRNAs were highly expressed in cultured cardiac fibroblasts. TGF-β1 (5 ng/mL) significantly increased Smad2 phosphorylation and [(3)H]-leucine incorporation, which were attenuated by pretreatment with efonidipine (10 µM). Neither R(-)efonidipine (10 µM), selective T-type CCB, nor nifedipine (10 µM), selective L-type CCB, efficaciously inhibited both TGF-β1-induced Smad2 phosphorylation and [(3)H]-leucine incorporation. However, both were markedly attenuated by combination of R(-)efonidipine and nifedipine, EDTA, or calcium-free medium. Pretreatment with Smad2 siRNA significantly attenuated [(3)H]-leucine incorporation induced by TGF-β1. These data suggest that efonidipine elicits inhibitory effects on TGF-β1- and Smad2-dependent protein synthesis through both T-type and L-type calcium channel-blocking actions in cardiac fibroblasts.

High Developmental Rates of Mouse Oocytes Cryopreserved by an Optimized Vitrification Protocol: The Effects of Cryoprotectants, Calcium and Cumulus Cells

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.

Gynura procumbens Merr. decreases blood pressure in rats by vasodilatation via inhibition of calcium channels

INTRODUCTION:Gynura procumbens has been shown to decrease blood pressure via inhibition of the angiotensin‐converting enzyme. However, other mechanisms that may contribute to the hypotensive effect have not been studied.OBJECTIVES:To investigate the cardiovascular effects of a butanolic fraction of Gynura procumbens in rats.METHODS:Anaesthetized rats were given intravenous bolus injections of butanolic fraction at doses of 2.5–20 mg/kg in vivo. The effect of butanolic fraction on vascular reactivity was recorded in isolated rat aortic rings in vitro.RESULTS:Intravenous administrations of butanolic fraction elicited significant (p<0.001) and dose‐dependent decreases in the mean arterial pressure. However, a significant (p<0.05) decrease in the heart rate was observed only at the higher doses (10 and 20 mg/kg). In isolated preparations of rat aortic rings, phenylephrine (1×10‐6 M)‐ or potassium chloride (8×10‐2 M)‐precontracted endothelium‐intact and ‐denuded tissue; butanolic fraction (1×10‐6–1×10‐1 g/ml) induced similar concentration‐dependent relaxation of the vessels. In the presence of 2.5×10‐3 and 5.0×10‐3 g/ml butanolic fraction, the contractions induced by phenylephrine (1×10‐9–3×10‐5 M) and potassium chloride (1×10‐2–8×10‐2 M) were significantly antagonized. The calcium‐induced vasocontractions (1×10‐4–1×10‐2 M) were antagonized by butanolic fraction concentration‐dependently in calcium‐free and high potassium (6×10‐2 M) medium, as well as in calcium‐ and potassium‐free medium containing 1×10‐6 M phenylephrine. However, the contractions induced by noradrenaline (1×10‐6 M) and caffeine (4.5×10‐2 M) were not affected by butanolic fraction.CONCLUSION:Butanolic fraction contains putative hypotensive compounds that appear to inhibit calcium influx via receptor‐operated and/or voltage‐dependent calcium channels to cause vasodilation and a consequent fall in blood pressure.

87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

N-Cadherin Expressing MM Cells Form Calcium-Dependent, Adherent Junctions That Likely Contribute to Focal Lesions and Nodular Growth of Multiple Myeloma Cells

AbstractAbstract 979E-cadherin-mediated adhesion regulates homeostasis in tissues of epithelial origin and homotypic N-cadherin interactions are central to the interaction of hematopoietic stem cells and the endosteal niche. Loss of E-cadherin in carcinomas is characteristic of the epithelial-to-mesenchymal transition and metastasis. The loss of E-cadherin, causes the release of b-catenin from the adherent complex, increased nuclear translocation, and increased transcriptional activity of b-catenin/TCF. We and others have demonstrated that alterations in the Wnt/b-catenin pathway exists in multiple myeloma, a malignancy of terminally differentiated antibody secreting plasma cells. The first evidence of this deregulation came from studies showing that MM cells secrete the potent Wnt/b-catenin signaling inhibitor DKK1. While it is now clear that DKK1 mediated suppression of Wnt/b-catenin in the bone marrow contributes to the decoupling of bone formation, the role of Wnt/b-catenin in normal plasma cell development and myelomagenesis is less clear and often controversial. Myeloma cells grow exclusively in the bone marrow. This growth is characterized as being interstitial or nodular, with most disease exhibiting a mixed pattern. Nodular growth, recognized as focal lesions (FL) on MRI, characterizes the conversion of MGUS to symptomatic MM. Consistently, DKK1 levels are highest in CD138 cells isolated from FL where nodular tumor growth and bone destruction may be linked by Wnt/b-catenin suppression in MM cells and local microenvironment. Given the central role of classical cadherins in promoting cell-cell adhesion and regulating b-catenin, we hypothesized that abnormal expression of cadherins might play a direct role in myelomagenesis.Cell lysates were prepared from 24 MM cell lines and from CD138+ cells from the BM of eight patients with MM. Immunoblotting was performed with antibodies specific to human N-cadherin protein. N-cadherin protein was observed in more than 82% of MM cell lines, with high protein levels in 55% of the cell lines. Similar levels of N-cadherin protein were seen in primary myeloma cells from the eight MM patients: two patients showed the highest levels of N-cadherin protein, two showed intermediate levels, and three weak levels; N-cadherin protein was absent in one patient. Similar results were obtained with three color-flow cytometry analysis of primary MM BM. To determine whether N-cadherin mediated MM cell interactions, a cell aggregation assay using GFP-expressing, N-cadherin+ MM cells observed under fluorescence microscopy was employed. When N-cadherin+ JJN3 cells were cultured in normal growth medium, they aggregated to form clusters. Similar results were observed in other MM cell lines, including OPM-2 and KMS-28-BM. Addition of a neutralizing N-cadherin antibody to these cultures significantly attenuated aggregation of JJN3 cells compared to control cells in normal medium or cells treated with control IgG. Homotypic N-cadherin interaction forms adherent junctions in a calcium dependent manner. To see if aggregation of MM cells results in the formation of adherent junctions, immunochemical staining was preformed to visualize the N-cadherin protein in OPM-2 cells. N-cadherin protein was clearly observed between myeloma cells in aggregation clusters that were significantly diminished in calcium-free medium. Taken together, these data suggest that N-cadherin induces homotypic adhesion of myeloma cells in a calcium-dependent manner and suggests that calcium release during bone resorption may enhance adherent junctions in MM that may in turn enhance plasma membrane localization of b-catenin. Studies are currently underway to determine whether DKK1 and N-cadherin adherent junctions cooperate to suppress b-catenin nuclear activity in MM cells and all converge to induce the nodular growth pattern and bone destruction often seen in N-cadherin-positive MM. Disclosures:No relevant conflicts of interest to declare.

Intracellular calcium as a second messenger following growth stimulation of human keratinocytes.

The mitogenic effect of the neuropeptide substance P and bombesin was investigated in normal human keratinocytes in serum-free culture, both with and without the presence of epidermal growth factor (EGF). Although both neuropeptides induced a small increase in cell numbers in the presence of EGF, the response was much greater in its absence, and cell numbers increased to 200% of controls at 5 days. Changes in intracellular free calcium are frequently seen following mitogenic stimulation of cells, and this phenomenon was studied in individual keratinocytes. Epidermal growth factor (10 ng/ml) induced calcium transients in 57% (n = 21) of cells. The mean intracellular free calcium was 97 +/- 11 nM (mean +/- SEM) in quiescent cells, and the calcium transients reached approximately 250 nM for 3-4 min. In the presence of EGF, calcium transients were never observed with the addition of either substance P or bombesin. For EGF-deprived cultures, 20% of keratinocytes (n = 10) showed a large calcium transient following the addition of 500 nM bombesin, and 63% (n = 12) of cells gave calcium transients following the addition of 700 nM of substance P. Studies in calcium-free medium, and following depletion of intracellular calcium stores with thapsigargin, showed that all of the calcium transients were dependent on the presence of intracellular stores, but also partially mediated by an influx of extracellular calcium. These studies demonstrate the mitogenic effect of substance P and bombesin on human keratinocytes in the absence of EGF. The ability of the neuropeptides to increase keratinocyte growth in culture suggests a possible in wound healing.