Calcineurin is required to release Xenopus egg extracts from meiotic M phase

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.     

Roles of protein kinase C in oocyte meiotic maturation and fertilization

Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.     

Irreversible swelling of secretory granules during exocytosis caused by calcium

The fusion of the limiting membrane of a secretory granule with the plasmalemma during exocytosis is equivalent to the fusion and release of contents that occurs when phospholipid vesicles fuse with planar bilayers. Experiments with bilayers demonstrate that phospholipid vesicles must swell if they are to fuse. Also, inhibition of exocytosis in solutions of high osmolarity occurs in several types of secretory cell. We report here experiments on the cortical granule exocytosis of sea-urchin eggs. Exocytosis is prevented when the osmolality of the medium surrounding the eggs is raised from 1 to 2 osmol kg-1. High osmolality also prevents calcium-dependent exocytosis in vitro. Prior treatment with calcium at high osmolality triggers fusion when normal osmolality is restored, even if calcium is removed before dilution. Addition of calcium causes the cortical granules to swell. The large increase in membrane capacitance which normally accompanies fusion is absent in eggs activated in solutions of high osmolarity. Our data are consistent with the idea that a secretory granule must swell to fuse with the plasma membrane and support the hypothesis of an osmotically driven fusion step during exocytosis.     

Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.     

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.     

Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations

The fertilization potential, or activation potential, has been demonstrated in the eggs of various species, and it has been shown to block polyspermy in echinoderm, echiuran and frog eggs, but no studies have been reported of electrical phenomena occurring when mammalian eggs are fertilized. We report here the fertilization potential of golden hamster eggs in vitro. To correlate the change of potential with the interaction between sperm and egg, only one sperm was attached to each egg. We found that a sperm induces recurring hyperpolarizations, constituting a fertilization potential which differs from that in the eggs of other species.     

NO is necessary and sufficient for egg activation at fertilization

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.     

中国对虾精子发生及受精细胞学

中国对虾（ＰｅｎａｅｕｓｃｈｉｎｅｎｓｉｓＯｓｂｅｃｋ１７６５）属于节肢动物门,有鳃亚门,甲壳纲,软甲亚纲,十足目,游泳亚目,对虾科,对虾亚科,对虾属,是我国最重要的经济虾类,主要分布在我国渤海、黄海,东海北部也有少量分布。常与墨吉对虾混栖。到目前为...     

Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs.

In vertebrates, mature eggs are arrested at the second meiotic metaphase by the cytostatic factor (CSF), now known to be the c-mos proto-oncogene product (Mos). Fertilization or egg activation triggers a transient increase in the cytoplasmic free calcium and releases the meiotic arrest by inactivating maturation/mitosis-promoting factor (MPF). CSF or Mos, which is also inactivated by the calcium transient, seems to stabilize MPF in mature eggs and CSF-injected embryos. Thus, it was assumed that CSF inactivation is the primary cause of MPF inactivation on meiotic release. We have directly compared the degradation kinetics of CSF (Mos) and MPF during meiotic release, using the same batch of Xenopus eggs. We report here that, at the molecular level, cyclin subunits of MPF are degraded before Mos is degraded and, at the physiological level, that MPF activity is inactivated before CSF activity during activation of Xenopus eggs. These results, in conjunction with circumstantial evidence, support the novel view that a calcium transient on fertilization induces a CSF-independent pathway for MPF inactivation, whereas CSF inactivation during meiotic release serves only to allow the fertilized egg to enter mitosis.     

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.     

Aggregation of chromaffin granules by calpactin at micromolar levels of calcium

Several cytosolic proteins bind to secretory granule membranes in a Ca2+-dependent manner and thus may be involved in the mediation of membrane interactions during exocytosis. One of these proteins, calpactin, is a tetramer consisting of two heavy chains of relative molecular mass (Mr) 36K (p36) and two light chains of 10K (p10). We report here that calpactin promotes the Ca2+-dependent aggregation and fatty acid-dependent fusion of chromaffin granule membranes at a level of Ca2+ that is lower than that reported for other granule-aggregating proteins, and which parallels the Ca2+ requirement for secretion from permeabilized chromaffin cells. We found subunits of calpactin to be inactive in promoting granule aggregation. Two distinct 33K proteolytic fragments of p36, differing at their N termini, also promote granule aggregation but with different Ca2+ sensitivities from calpactin. These differences suggest that the N-terminal portion of p36 modulates the Ca2+/lipid binding sites in the core portion of p36 (ref.5).     

Calcium/calmodulin-dependent protein kinase II increases glutamate and noradrenaline release from synaptosomes

A variety of evidence indicates that calcium-dependent protein phosphorylation modulates the release of neurotransmitter from nerve terminals. For instance, the injection of rat calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-dependent PK II) into the preterminal digit of the squid giant synapse leads to an increase in the release of a so-far unidentified neurotransmitter induced by presynaptic depolarization. But until now, it has not been demonstrated that Ca2+/CaM-dependent PK II can also regulate neurotransmitter release in the vertebrate nervous system. Here we report that the introduction of Ca2+/CaM-dependent PK II, autoactivated by thiophosphorylation, into rat brain synaptosomes (isolated nerve terminals) increases the initial rate of induced release of two neurotransmitters, glutamate and noradrenaline. We also show that introduction of a selective peptidergic inhibitor of Ca2+/CaM-dependent PK II inhibits the initial rate of induced glutamate release. These results support the hypothesis that activation of Ca2+/CaM-dependent PK II in the nerve terminal removes a constraint on neurotransmitter release.     

斑马鱼受精过程中原核的时空规律

斑马鱼的成熟卵子处于第2次减数分裂中期,精子通过受精孔进入卵内,精子入卵后,卵子被激活;受精后3 min,精核位于卵质膜内侧边缘,处于凝集状态;受精后5 min精核开始解凝;受精后8 min,精核明显解凝,受精卵出现明显的精子星光.同时进入第2次减数分裂后期,之后排出第二极体;受精后10 min,雄原核形成,具有双星光,此时,雄原核已位于胚盘中部;受精后15 min雌原核已形成,雌、雄原核向胚盘中央靠近;受精后20 min,雌雄原核开始接触;受精后30 min,雌雄原核完全融合成为1个合子核;35 min时,受精卵开始第1次卵裂;45 min后分裂形成2个子核;50 min时分裂为2细胞,60 min时分裂为4细胞.     

An extrachromosomal factor causing loss of paternal chromosomes

Extrachromosomal inheritance is ubiquitous among plants and animals; however, most extrachromosomal factors are uniparentally inherited through females, but not through males. Examples include chloroplasts, mitochondria and a variety of intracellular symbionts. The only known exception to maternal extrachromosomal inheritance in an animal is a paternally transmitted sex ratio factor (psr) which causes all-male families in the parasitic wasp, Nasonia vitripennis. Normally in this wasp, male offspring are haploid and develop from unfertilized eggs whereas females are diploid and develop from fertilized eggs. The psr factor is either a venereally transmitted infection which prevents egg fertilization (and therefore causes all-male families), or a factor transmitted to eggs by the sperm of males carrying psr, which somehow prevents incorporation of the paternal chromosomes. Here we report that sperm from psr males fertilizes eggs, but that the paternal chromosomes are subsequently condensed into a chromatin mass before the first mitotic division of the egg and do not participate in further divisions. Resulting haploid offspring are male, but have inherited the paternal factor. This extrachromosomal factor promotes its own transmission at the expense of the paternal chromosomes, and therefore can be considered a 'selfish' genetic element.     

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.