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1.
    
Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an IC50 of 7.5 x 10(-3) MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K+ loss simultaneously with a Na+ entry into unfertilized eggs and increased the Ca2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATP-dependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca2+ from a preparation pre-loaded with 45Ca2+. Verapamil (10(-4) M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellular compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin.  相似文献   

2.
The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K+] sea water were examined. In high [K+] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K+ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K+ sea water. However, in high [K+] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca2+ concentration of spermatozoa in normal sea water but not in 100mM K+ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K+] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K+ channels.  相似文献   

3.
    
Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca2+ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca2+ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca2+ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca2+ channels mediate the intracellular Ca2+ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.  相似文献   

4.
Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.  相似文献   

5.
Mitochondria isolated from sea urchin embryos in early development show almost the same activities of cytochrome c oxidase and flavin-linked complex enzymes, which are estimated by cytochrome c reductases as in those isolated from unfertilized eggs. The activities of these cytochrome c reductases are inhibited by Ca2+ at above 10-5 M more strongly than cytochrome c oxidase. To investigate the changes in intramitochondrial Ca2+ concentration at fertilization, the activity of pyruvate dehydrogenase, another mitochondrial enzyme, was measured. The activity of this enzyme was controlled by phosphorylation and Ca2+-dependent dephosphorylation of the catalytic unit. The enzyme activity increased for 30 min after fertilization, decreased and became close to zero within ~60 min. Then, the activity appreciably increased again after hatching. This seems to reflect changes in the intramitochondrial Ca2+ concentration. The enzyme activity was enhanced by pre-incubation with Ca2+ at concentrations up to 10-5 M but was made quite low at above 10-4 M Ca2+ and 10-3 M adenosine triphosphate. Although the changes in pyruvate dehydrogenase activity observed at fertilization will reflect the changes in the intramitochondrial calcium concentration, the intramitochondrial Ca2+ concentration of unfertilized eggs cannot be estimated from these results because high (> 10-4 M) or low (10-6 M) Ca2+ can inhibit the enzyme. Measurement of respiration of a single egg showed that injection of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid released the mitochondrial electron transport in the unfertilized egg. The possibility that changes in intramitochondrial calcium concentration occur at fertilization is discussed in relation to activation of both mitochondrial respiration and pyruvate dehydrogenase.  相似文献   

6.
    
Danilo Guerini 《Biometals》1998,11(4):319-330
The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers  相似文献   

7.
Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.  相似文献   

8.
    
In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca2+]i (the intracellular free Ca2+ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca2+]i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca2+]i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca2+-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca2+]i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca2+-independent reaction downstream of Ca2+-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.  相似文献   

9.
The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.  相似文献   

10.
    
Summary— Sea urchin embryos can be easily dissociated into single cells by exposure to Ca2+- and Mg2+-free seawater. When transferred back to normal seawater, isolated cells spontaneously form aggregates capable of development. Here, the Ca2+-dependent self-aggregation of toposome, a 22S glycoprotein complex which mediates cell-cell adhesion in sea urchin embryos, has been investigated using the purified molecule. Results show that the 22S complex is completely converted to 15S particles by sedimentation on sucrose isokinetic gradients in the presence of EDTA. Reconstitution of the 22S complex is achieved by readdition of Ca2+. We propose that the 15S particle constitutes the toposome functional unit on the cell surface.  相似文献   

11.
Calcium in fungi   总被引:2,自引:0,他引:2  
Abstract. Recently much experimental evidence has accumulated concerning intracellular calcium and its fundamental role as a regulator in eukaryotic cells. The literature relating to Ca2+ in fungi is large and diverse and this paper draws together the available information and discusses the particular functions of the ion in this group of organisms.
Uptake mechanisms in fungi are considered with special reference to the effect of Ca2+ on permeability and the systems responsible for transport of ions, sugars and amino acids. Discussion of the subcellular locations and distribution of Ca2+ is accompanied by a critique of methodology used in determination of subcellular sites of Ca2+ in fungi. The role of Ca2+ in morphogenesis in fungi is considered with particular reference to selected groups.  相似文献   

12.
    
Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.  相似文献   

13.
    
Sperm centrosomal function was assessed by immunocytochemical analysis after the injection of human sperm into mature rabbit eggs. Three hours after intracytoplasmic sperm injection (ICSI), an astral microtubule array from the base of the human sperm was observed in the rabbit eggs. This sperm aster expanded in the egg cytoplasm, concomitant with pronuclear formation, and a dense microtubule array was organized at the time of pronuclear centration. Using fertile donor sperm, the sperm aster formation rate at 3 hr after ICSI was 35.0 +/- 1.5%. Using sperm from infertile patients, the average aster formation rate was lower (25.4 +/- 14.8%, P<0.05). Among infertile cases, there was no correlation between sperm aster formation rates and conventional parameters of semen analysis. However, the sperm aster formation rate correlated with the embryonic cleavage rate following human in vitro fertilization (IVF). These data suggest that this assay reflects sperm function during embryonic development after sperm entry and that reproductive success during the first cell cycle requires a functional sperm centrosome. Furthermore, sperm centrosomal function cannot be predicted from conventional parameters of semen analysis. We propose that insufficient centrosomal function could be the cause of certain cases of idiopathic infertility. These assays may lead to the discovery of new types of infertility, which have previously been treated as \"unexplained infertility,\" and may also lead to the treatment of infertility incurable even by ICSI. Consequently, an accurate and relevant assay to help assure couples of the success of fertilization is warranted, perhaps prior to ICSI therapy.  相似文献   

14.
    
Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca2+]i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca2+]i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward‐rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S‐type anion channels and down‐regulate inward‐rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.  相似文献   

15.
胍丁胺对大鼠心室肌细胞内游离钙浓度的影响   总被引:1,自引:1,他引:1  
Li Q  Shang ZL  Yin JX  Wang YH  He RR 《生理学报》2002,54(6):467-472
本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关  相似文献   

16.
三羟异黄酮对豚鼠心室肌细胞内游离钙浓度的影响   总被引:3,自引:0,他引:3  
Ji ES  Wang C  He RR 《生理学报》2004,56(2):204-209
用激光共聚焦显微镜观察研究三羟异黄酮(genistein,GST)对豚鼠心室肌细胞内游离钙浓度([Ca^2 ]i)的影响。结果用相对荧光强度(FI-F0/FX0,%)表示。实验结果显示,在正常台氏液、无钙台氏液和正常台氏液中加入3mmol/L EGTA后,GST(10~40μmol/L)浓度依赖性地降低细胞内钙浓度。蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate)和L-型Ca^2 通道激动剂Bay K8644可部分抑制正常台氏液时GST的效应。当细胞外液钙浓度由1mmol/L增加到10mmol/L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,GST(40μmol/L)可降低钙波的传播速度和持续时间,最终阻断钙波。以上结果提示,GST降低心室肌细胞内游离钙浓度,此作用与其抑制电压依赖性Ca^2 通道、减弱酪氨酸激酶抑制和豚鼠心室肌细胞肌浆网内钙释放有关。  相似文献   

17.
Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.  相似文献   

18.
The effect of 2 mM AlCl3 on NAD+ kinase (E.C. 2.7.1.23) activity was studied using Euglena gracilis strain Z grown heterotrophically in darkness at pH 3.5 in the presence of lactate as sole carbon source. The Al-treatment slowed down the culture growth and suppressed the peak of NAD+ kinase activity, which characterizes the beginning of the exponential phase of growth of the control cell cultures. There are two possible explanations of the Al effect: it 1) either prevents the enzyme activation by the Ca-calmodulin (CaM) complex; or 2) suppresses the CaM-dependent NAD+ kinase form. In Euglena cells, a part of the NAD+ kinase activity is enhanced by EGTA and lowered by Ca2+: this peculiar NAD+ kinase activity is unaffected by the Al treatment. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

19.
The oxidation of exogenous NADH by Jerusalem artichoke ( Helianthus tuberosus L.) tuber mitochondria was strongly inhibited at pH 7.2 by EDTA, EGTA and mersalyl and by chlorotetracycline in the presence of Ca2+. This inhibition disappeared at pH 5.5 where about 50% activity was found as compared to controls at pH 7.2. The rate of oxidation of NADPH at pH 5.5 was the same as for NADH but it was inhibited by 50% by both EDTA and mersalyl.
Mitochondria from Arum maculatum spadices oxidised NADH and NADPH with pH optima of 7.2 and 6.5, respectively. In the presence of EDTA the optima shifted to 6.7 and 5.9, respectively, due to an inhibition at higher pH and a lack of inhibition at lower pH. At pH 6.7 NADH oxidation was completely insensitive to both EDTA and mersalyl whereas the oxidation of NADPH was inhibited by more than 50%. The inhibition of NAD(P)H oxidation by chelators at neutral pH was due to the removal of Ca2+ from the membranes in both types of mitochondria. The differences observed in the properties of NADH and NADPH oxidation suggest that two different dehydrogenases are involved. Because of the strong pH-dependence and the changes in chelator-sensitivity in the physiological pH-range 6–8 it is suggested that the properties of NAD(P)H oxidation provide the cell with important means of metabolic regulation.  相似文献   

20.
    
The apical meristem of the short‐day plant Chenopodium rubrum responds to photoperiodic flower induction with specific changes of pH and Ca2+ patterning immediately after the inductive dark span. The red–far‐red reversibility of the pH and Ca2+ patterning in response to night break treatments was measured in order to distinguish between the effect of the prolonged dark span per se and the specific effect of photoperiodic flower induction. In addition, the pH and Ca2+ patterning in C. rubrum was compared with the long‐day plant Chenopodium murale. The pH was visualized using the fluorescent probe carboxy SNARF‐1. Calcium ion concentrations were studied using a combination of Ca2+‐probes Fluo‐3 and Fura Red. It was observed that the specific changes in pH and Ca2+ patterning at the apical meristem of C. rubrum were abolished by the red‐light break. This effect was fully reversed with a subsequent single far‐red treatment. These observations infer the influence of phytochrome on both pH and Ca2+ patterning. Changes in pH and Ca2+ patterning upon flower induction were observed in both long‐day and short‐day plants. These results support the hypothesis that changes of pH and [Ca2+] in cells of the apical meristem are part of the pathway in signal transduction triggering flower initiation.  相似文献   

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