Activation of porcine oocytes using cyclopiazonic acid, an inhibitor of calcium-dependent ATPases

Cyclopiazonic acid (CPA), a potent inhibitor of endogenous calcium-dependent ATPases, is able to induce parthenogenetic activation in pig oocytes matured in vitro. Sixty-four percent of matured pig eggs cultured with 100 nM CPA for 4 hr were activated. A similar activation rate was observed in oocytes treated with thapsigargin, another inhibitor of calcium-dependent ATPases. The parthenogenetic development of CPA-activated eggs did not proceed beyond the 8-cell stage. The blockage of calcium channels by verapamil only slightly decreased the proportion of CPA-activated pig oocytes. This indicates that the release of calcium from intracellular stores is sufficient for oocyte activation and calcium influx from extracellular sources has no significant role. The significant decrease in CPA-activated oocytes (100 nM of CPA for 4 hr) after a microinjection of heparin indicated that the mobilization of intracellular calcium stores is mediated through inositol trisphosphate receptors. On the other hand, the only slightly depressed activation rate in oocytes microinjected with ruthenium red and procaine indicates that CPA mobilizes a much smaller amount of calcium through the ryanodine receptors. The marked inhibitory effect of ophiobolin A and W7 on the activation of CPA-treated pig oocytes suggests that the calcium signal, as the second messenger, acts downstream through calmodulin. J. Exp. Zool. 287:304-315, 2000.     

Influence of ryanodine and inositol trisphosphate receptors inhibitions on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP

The influence of ryanodine and inositol triphosphate receptors inhibitors on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP was investigated using fluorescent dye chlortetracycline. Porcine oocytes were isolated from ovaries with yellow body. Ca2+ exit from intracellular stores of porcine oocytes activated by prolactin (5 and 50 ng/ml) in calcium free medium was decreased after treatment of oocytes by heparin (inhibitor of inositol triphosphate receptors) and was not changed after treatment of oocytes by ruthenium red (inhibitor of ryanodine receptors). Inhibition of protein kinase C did not affect on the Ca2+ exit stimulated by prolactin. GTP did not stimulate Ca2+ exit from intracellular stores of pig oocytes, and inhibitors of both calcium channels and proteinkinase C had no influence on this process. The joint action of prolactin and GTP did not result in additional Ca2+ exit from intracellular stores of oocytes after both pretreatment and untreatment by the inhibitor of protein kinase C. The data obtained testify to activation of IP3-sensitive receptors under effect of prolactin and in the absence of GTP influence on these receptors.     

Dynamic changes to the inositol 1,4,5-trisphosphate and ryanodine receptors during maturation of bovine oocytes

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyR) have been identified as two ligand-gated calcium channels which play a critical role in mediating calcium release in many different types of cells and tissues. The physiological significance of the two receptors in regulation of intracellular calcium during meiotic maturation and fertilization in the bovine oocyte was evaluated. Metabolic labeling of bovine oocytes by Met-Cys 35S during early and late maturation was followed by immunoprecipitation of both RyR and IP3R using specific antibodies against these two receptors. Results indicate that IP3R is translated throughout the maturation period; in contrast, RyR is only translated during the late maturation period of bovine oocytes. In addition, the experiments reported here investigate the temporal and spatial relationships between these calcium channels and the endoplasmic reticulum (ER) and cortical granules (CG). Immunocytochemistry, fluorescence staining and confocal microscopy were applied at four oocyte developmental stages: the germinal vesicleintact (GV-intact), metaphase I (MI) and metaphase II (MII) stages of maturation and the fertilized egg at 6 h post insemination (hpi). Although oocytes demonstrated some differences in staining patterns and localization, both receptor types showed apparent dynamic changes during meiotic maturation and dramatic decreases in signals after insemination. These results indicate the changes in the number and distribution of IP3R and RyR may account for the increased intracellular calcium responsiveness at fertilization. The IP3R appears to associate with the ER at the sub-vitelline membrane cortex in bovine oocytes. In addition, RyR appears to associate with the CG. In conclusion, although these two receptors may have different functional roles in regulation of calcium release during meiotic maturation and fertilization, it appears that both IP3R and RyR contribute to the significant increase of intracellular calcium during fertilization and activation in the bovine oocyte.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

IP3-dependent calcium-induced calcium release mediates bidirectional calcium waves in neurones: functional implications for synaptic plasticity

IP(3)-dependent calcium-induced calcium release (ICICR) is a general mechanism of calcium release that occurs in pyramidal neurones of hippocampus, the neocortex and in Purkinje cells of the cerebellar cortex. When ICICR is initiated synaptically in dendrites of neurones from brain slices, calcium waves can propagate bidirectionally to the soma and distal dendrites. ICICR relies on the coincidence of a calcium influx triggered by the backpropagation of action potentials and the activation of cholinergic, serotoninergic or glutamatergic metabotropic receptors. The involvement of IP(3) receptors (IP(3)R) in ICICR is clearly established. In contrast, ryanodine receptors (RyR) do not seem necessary for the triggering and propagation of calcium waves, but ICICR depends on calcium stores sensitive to ryanodine. Thus, the role of RyR remains to be established. ICICR provides a mechanism for global calcium signalling in neurones that may be involved in the reinforcement of Hebbian plasticity, heterosynaptic plasticity and cell death.     

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

Stores not just for storage. intracellular calcium release and synaptic plasticity.

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.     

Focal sarcoplasmic reticulum calcium stores and diffuse inositol 1,4,5-trisphosphate and ryanodine receptors in human myometrium

Intracellular calcium stores of human uterine myocytes in primary and second passage cell culture were visualized using the low-affinity calcium-sensitive fluorescent dye, fluo-3FF. The calcium stores appeared as numerous small (0.2-0.5 microm diameter) focal fluorescences. The stores were not depleted by exposing the cells to oxytocin or ryanodine under standard conditions. The stores were rapidly depleted by oxytocin or ryanodine exposure when sarcoplasmic reticulum (SR) calcium re-uptake was inhibited by pretreatment with thapsigargin. Immunofluorescence experiments indicated that both ryanodine and inositol 1,4,5-trisphosphate (IP(3)) receptors were smoothly distributed throughout the SR, and neither receptor co-localized with the calcium stores. Since IP(3) and ryanodine calcium channels are tightly associated with their receptor, these results suggest that SR calcium release occurs via second messenger channels that are remote from the SR calcium stores. These observations are consistent only with a mechanism for release of calcium stores where the SR serves three functions: (1) as site of calcium storage, (2) as the structure that contains the IP(3)- and ryanodine receptors and their associated release channels, and (3) as a conduit between the calcium stores and the release channels.     

Gas6在猪卵母细胞及早期胚胎中表达模式与功能的初步研究

该研究通过免疫组化和QRT-PCR等方法系统分析了Gas6（growth arrest-special gene 6）基因在猪卵泡发生及早期胚胎发育过程中的表达规律,并提出了一种改良猪卵母细胞体外成熟系统的方法。研究结果显示,Gas6基因表达于猪卵巢中的卵母细胞细胞核及其周围的卵丘细胞,在卵母细胞体外成熟及早期胚胎发育过程中始终有表达,且在囊胚中表达最高。Gas6 mRNA在卵母细胞成熟过程中始终存在,但在孤雌激活后迅速消失,直到发育到囊胚时再次出现。在猪卵母细胞体外培养系统中添加不同浓度的Gas6重组蛋白培养卵母细胞,发现添加Gas6重组蛋白对卵母细胞的极体率无显著影响;但是当添加浓度为100 ng/mL时,培养的卵母细胞孤雌激活后分裂率、囊胚率及囊胚细胞数都显著增高。Gas6可能是通过改善卵母细胞细胞质的成熟质量提高卵母细胞的发育潜能,从而获得了更多、更好的胚胎。     

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).     

Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation

Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP?Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP?Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP?Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP?Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP?Rs, probably through a CICR mechanism. Both receptors play a role in Ca2+ release mechanisms although their relative contribution to the activation process is unclear.     

In vitro ageing of pig oocytes: effects of the histone deacetylase inhibitor trichostatin A.

After in vitro maturation, the unfertilized pig oocytes underwent the process called ageing. This process involves typical events such as fragmentation, spontaneous parthenogenetic activation or lysis. Inhibition of histone deacetylase, using its specific inhibitor trichostatin A (TSA), significantly delayed the maturation of pig oocytes cultured in vitro. The ageing of oocytes matured under the effect of TSA is the same as the ageing in oocytes matured without TSA. The inhibition of histone deacetylase during oocyte ageing significantly reduced the percentage of fragmented oocytes (from 30% in untreated oocytes to 9% in oocytes aged under the effect of 100 nM of TSA). Oocytes matured in vitro and subsequently aged for 1 day under the effects of TSA retained their developmental capacity. After parthenogenetic activation, a significantly higher portion (27% vs. 15%) of oocytes developed to the blastocyst stage after 24 h ageing under 100 nM TSA when compared with oocytes activated after 24 h ageing in a TSA-free medium. The parthenogenetic development in oocytes aged under TSA treatment is similar to the development of fresh oocytes (29% of blastocyst) artificially activated immediately after in vitro maturation.     

Activation of pig oocytes using nitric oxide donors

Nitric oxide (NO) plays an important role in intracellular signaling, but its role during the activation of mammalian oocytes is little understood. In our study, in vitro matured pig oocytes were cultured with NO-donors-S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitropruside (SNP). These treatments were able to induce parthenogenetic activation of pig oocytes matured in vitro. The specificity of this effect was confirmed by the activation of oocytes by exogenous endothelial nitric oxide synthase (eNOS) microinjected in the oocyte with its activator calmodulin. Relatively long exposure (10 hr) is needed for activation of pig oocytes with 2.0 mM SNAP. An active NOS is necessary for the NO-dependent activation of pig oocytes because NOS inhibitors L-NMMA or L-NAME are able to inhibit activation of oocytes with NO-donor SNAP. On the basis of our data, we conclude that the NO-dependent activating stimulus seems inadequate because it did not induce the exocytosis of cortical granules. Also, the cleavage of parthenogenetic embryos was very low, and embryos did not develop beyond the stage of eight blastomeres.     

Regulation of ryanodine receptor opening by lumenal Ca(2+) underlies quantal Ca(2+) release in PC12 cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.     

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.     

Calcium release of heat-shocked porcine oocytes induced by thimerosal or inositol 1,4,5-trisphosphate (IP3)

The objective of this study was to determine the effect of heat shock (HS) on the Ca(2+) release and the subsequent development in matured porcine oocytes. Oocytes were matured in vitro and randomly allocated to different heat treatments at 41.5 degrees C for 1 (HS1h), 2 (HS2h) or 4h (HS4h). Control groups of oocytes were cultured for 0 or 4h without HS (39 degrees C, C0h, C4h). In Experiment 1 (eight replicates), matured oocytes were activated by thimerosal (200 microM, 10 min) following HS. Among all heated groups, maximal intracellular calcium concentration ([Ca(2+)](i)) was the highest in the HS2h. The lowest [Ca(2+)](i) peak among HS groups was observed in the HS4h, but it was higher than that in the non-heated C4h group (P<0.05). In Experiment 2 (12 replicates), each matured oocyte was injected with IP(3) (0.5mM) and the Ca(2+) transient was recorded. The peak [Ca(2+)](i) in the C4h group was still the lowest among all groups (P<0.05). Total Ca(2+) release in HS2h appeared the highest among all treatments, and it was significantly higher than that in HS1h and C4h groups (P<0.05). In order to clarify the effect of incubation time in vitro (Experiment 3), matured oocytes were cultured at 39 degrees C for 0, 2 and 4h prior to treatment with thimerosal or injected with IP(3) (three replicates). The Ca(2+) release of matured oocytes declined with the prolonged culture (P<0.05). Finally, the development of HS-oocytes was evaluated after parthenogenetic activation (Experiment 4, three replicates), and the proportion of embryos developing to the blastocysts were lower (P<0.05) in the HS groups (31+/-7% to 33+/-1%) than in the control groups (52+/-11% to 56+/-9%). We conclude that HS alters the Ca(2+)-releasing ability of matured pig oocytes, and that heat-shocked oocytes with greater Ca(2+) release incur a low developmental competence after parthenogenetic activation.