Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions including meiotic maturation and parthenogenetic activation of mammalian oocytes. We observed that nitric oxide donor SNAP was potent to induce parthenogenetic activation in Xenopus eggs. NO-scavenger CPTIO impaired the effects of SNAP, providing evidence for the effects of the latter to be specific upon NO release. In Xenopus eggs, SNAP treatment induced pigment rearrangement, pronucleus formation and exocytosis of cortical granules. At a biochemical level, SNAP exposure lead to MAPK and Rsk inactivation within 30 minutes whereas MPF remained active, in contrast to calcium ionophore control where MPF activity dropped rapidly. MAPK inactivation could be correlated to pronuclear envelope reformation observed. In SNAP-treated eggs, a strong increase in intracellular calcium level was observed. NO effects were impaired in calcium-free or calcium limited medium, suggesting that that parthenogenetic activation of Xenopus oocytes with a NO donor was mainly calcium-dependent.
Citation: Jeseta M, Marin M, Tichovska H, Melicharova P, Cailliau-Maggio K, Martoriati A, et al. (2012) Nitric Oxide-Donor SNAP Induces Xenopus Eggs Activation. PLoS ONE 7(7): e41509. https://doi.org/10.1371/journal.pone.0041509
Editor: Reiner Albert Veitia, Institut Jacques Monod, France
Received: February 7, 2012; Accepted: June 22, 2012; Published: July 23, 2012
Copyright: © 2012 Jeseta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the Czech Science Fundation (GACR 523/08/H064), the ministry of education youth and sports of Czech Republic (MSM 6046070901), the Czech ministry of agriculture (MZe 0002716202) and Econet grant 18857PA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Nitric oxide (NO) is produced by Nitric Oxide Synthase (NOS) from L-Arginine and molecular oxygen, through a process that can also generate L-citruline . NOS is expressed in three isoforms, which are homodimers , . Constitutional isoforms of NOS, neuronal NOS (nNOS) and endothelial NOS (eNOS), are calcium and calmodulin-dependent and produce small amounts of NO for a short time lapse , . An inducible isoform of NOS (iNOS) drives sustained NO production , , which is independent on calcium or calmodulin . NOS isoforms have been detected in many cellular type . Particularly, NOS have been isolated from a variety of mammalian reproductive tissues like ovary, uterus, testis or epididymis; the role of NO has been emphasized in many physiological processes including reproduction , . NO was involved for example in the regulation of follicle growth and ovulation in mouse , spermatogenesis in human , embryo implantation in rats  and meiosis in pig and mouse , . The data collected in the different above-mentioned species suggest that NO might have a widely role in reproduction through mechanisms conserved through evolution but one cannot discard that NO effects may be also dependent upon the species considered.
Meiosis is a mode of cell division in which a diploid cell undergoes through two successive divisions without replication, to produce haploid cells, namely oocytes and polar bodies in case of female gametes. Vertebrate oocyte is arrested in prophase of the first meiotic division (MI), resume meiosis in response to hormonal stimulation, in the process called maturation, and are stopped at metaphase of the second division, in anticipation to fertilization . Metaphase II arrest is due to a cytostatic factor (CSF), whose function is to maintain high level of active MPF (M-Phase Promoting Factor ) within the cells. MPF promotes M-phase entry during mitosis or meiosis, and is made up of a catalytic subunit, Cdk1, and a regulatory sub-unit, Cyclin B . The activity of this heterodimer is regulated by inhibitory phosphorylation on Thr14 and Tyr15, achieved by Wee1 and Myt1 kinases  and Cyclin B level, which can be degraded through the ubiquitin pathway . Maintenance in metaphase II arrest is then achieved by preventing Cyclin B degradation. Though the nature of CSF has remained elusive for decades, it involved the Mitogen-Activated Protein Kinase (MAPK) cascade, whose components are involved in the prevention of Cyclin B degradation, and Emi2, which acts as an inhibitor of an Ubiquitin Ligase responsible from the metaphase-anaphase transition, the Anaphase Promoting Complex (APC) –.
Maintenance of MPF activity in metaphase II-arrested oocytes, is broken by the interaction of sperm with the egg membrane, which leads to the activation of the egg metabolism and meiosis completion . These cellular events, which are prepared by the cellular differentiation period related to maturation, also allow the transition from the eggs to the embryos . They include the onset of the different polyspermy blocks, the completion of meiosis and the onset of embryonic mitosis –. Completion of meiosis is attested by the extrusion of the second polar body and pronucleus formation. Following fertilization as well as following parthenogenetic activation, MPF drops within five minutes while MAPK remains active for 30 minutes before decreasing , . Inactivation of MPF is mainly due to the degragation of the Cyclin B whereas inactivation of MAPK reflects the degradation of its upstream activator Mos . Exit from metaphase II could be mediated by a transient or sustained increase in intracellular calcium, depending on the considered species. In Xenopus laevis, the egg activation is mediated by a rise in intracellular calcium concentration with specialized spatial and temporal dynamics , and CaMKII is considered as the key protein in MPF down-regulation, through promoting Emi2 degradation and APC activation . The specialized fertilization-specific calcium signal is shaped like a slow sweeping wave followed by a high calcium plateau that lasts for several minutes –. In non-excitable cells in general and in Xenopus oocyte particularly, an increase of intracellular calcium concentration could come both from release of calcium from internal stores (reticular or/and mitonchodrial stores) and influx from the external medium through calcium channels. In response to intracellular calcium stores depletion (i.e. rise of cytosolic calcium concentration), calcium enters the cells through calcium channels from the outside. The mechanism is called Store-Operated Calcium Entry (SOCE, through SOC channels) also known as capacitative calcium entry . It is to note that in metaphase II-arrested oocytes SOCE could not be activated any more as a consequence of MPF activation , , . Therefore, the calcium signalling pathways associated with oocyte egg activation is generated without any contribution of SOCE channels. Exit from metaphase II can be mimicked or stimulated by the calcium ionophore A23187 or by kinase inhibitors , . As described and reviewed in , the maturation process implies numerous reorganizations of calcium signaling actors such as internalization of plasma membrane calcium ATPase (PMCA) or expression at the plasma membrane of calcium-activated chloride channels to prevent polyspermy.
(A) Typical morphologies of Xenopus laevis oocytes in control batches, arrested in metaphase II, (B) following parthenogenetic activation by A23187 treatment or (C) following SNAP treatment. Scale bars represent 200 µm. (D) SNAP treatment induces release of cortical granule lectins from Xenopus oocytes. Oocytes were incubated for 1 hour without or with 5 mM SNAP or 10 µM A23187. After 1 hour the fluid surrounding oocytes (15 µl) was collected for analysis by SDS-PAGE and SYPRO Ruby staining. Molecular weight standards are indicated in kDa.
Nitric oxide has been considered as a potential regulator of meiosis , . Free radical nitric oxide was first reported to trigger parthenogenetic activation in sea urchin oocytes and suggested as a potential physiological regulator for egg activation . This proposal has been shaded by the fact that nitric oxide did not appear as a key factor for fertilization in mice and ascidians. Sperm or sperm extract injections induce strong calcium response without affecting nitric oxide level . Nevertheless, nitric oxide donors induce increase of intracellular levels of free calcium in mouse, ascidian  and sea urchin eggs , suggesting NO potentially acts through calcium mobilization. In porcine oocytes, NO donors were explored as potent parthenogenetic inducers , and egg activations were prevented by use of BAPTA-calcium chelator . Then, NO is rather not a primary stimulus for oocyte activation, though it drives parthenogenesis through calcium mobilization in this mammalian model. However in porcine oocytes cumulus cells around oocyte play an important role in supporting maturation and the gap junctions between cumulus cells and oocyte enable transport of small molecules regulating meiotic maturation . In previous reports were presented differences between DO (denuded oocytes) and COC (cumulus oocyte complexes) in various effect of NOS inhibitors on meiotic maturation in pig or mouse oocytes , . Then, it appeared difficult to discriminate if the effects of NO donors are due to effects on follicular or on oocytes itselves.
Our current understanding of meiosis regulation in vertebrate oocytes largely benefited from studies performed in amphibian models such as Xenopus laevis. Oocytes from this animal model offer several advantages including year-around availability, cell cycle synchronicity and ease of amenability for manipulation and large amount of protein, enabling biochemical studies on 1/10th of cells. Noticeably, oocytes resume meiosis under progesterone stimulation, in a process that is totally independent of follicular cells. Therefore, the present study aimed at evaluating the effect of nitric oxide donors on metaphase II block in Xenopus laevis oocytes. We tested the potential effects of NO donors on the activation of Xenopus laevis oocytes and thereby determine their effect on cortical exocytosis, cortical reaction, pronuclear formation. Here we report for the first time a parthenogenetic activation induced by NO donor in aquatic vertebrates - amphibian: nitric oxide donor - SNAP (S-nitroso-N-acetyl-DL-penicillamine) drives an atypical parthenogenetic activation of Xenopus eggs where MAPK cascade is broken in absence of MPF inactivation. Dependency upon Ca2+-dependent mechanisms is discussed.
All animal experiments were performed at the animal facility of the USTL Lille according to the rules of the European Community Council guidelines (86/609/EEC) for laboratory animal experimentation. The animal protocol was approved by the local institutional review board (Comité d’Ethique en Expérimentation Animale Nord-Pas-De-Calais, CEEA 07/2010).
(A) Kinetic of parthenogenetic activation after SNAP treatment. Matured oocytes were cultured with 5 mM SNAP or 10 µM A23187; cortical reaction was scored at 10 minutes time intervals. Data are shown as mean percentage of activation ± SEM of five independent experiments. (B) Western blot analysis. Each time 5 oocytes were taken off, homogenized in lysis buffer, and immunoblotted with antibodies against Cyclin B, P-Tyr15 Cdk1, Xp42Mpk1 and p90Rsk. (C) Western blot analysis. Neither Cyclin B or Mos were degraded in SNAP-treated matured oocytes (both were still detected 70 min after the beginning of the treatment); MPF activity was attested by its ability to phosphorylate histone H3. It is to note that Cdk1 remained unphosphorylated at tyrosine 15. (D) Immunoprecipitation. Cdk1 and Cyclin B are complexed together i metaphase II arrested oocytes. SNAP did not induce any separation between the two partners of the MPF heterodimer, even after 70 minutes.
Handling of Frogs and Oocytes
After anesthetizing Xenopus females (purchased from the University of Rennes I, France) by immersion in 1 g/l MS222 solution (tricaine methane sulfonate; Sandoz), ovarian lobes were surgically removed and placed in ND96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes–NaOH, pH 7.5). Fully grown stage VI oocytes were isolated and defolliculated by partial collagenase treatment for 30 min (1 mg/ml collagenase A, Roche Applied Science) followed by a manual microdissection. Oocytes were stored at 14°C in ND96 medium until experiments. Meiotic resumption was induced by incubation of oocytes at 19°C in ND96 medium containing 10 µM of progesterone (Sigma–Aldrich). Maturation process (or M-Phase entry) was scored by the appearance of a white spot at the animal pole of the oocyte. Activation was scored by occurence of pigment rearrangement typical of cortical reaction.
(A) Effect of CPTIO on meiotic maturation. Histogram showing percentages of matured oocytes (with white spot) after overnight treatment with progesterone (10 µM) either in medium ND96 supplemented 10 mM CPTIO (CPTIO) or oocyte in pure ND96 injected 40 mM CPTIO (injCPTIO). Groups PG + (only progesterone), PG − (without progesterone) and inj water (progesterone and injection 15nl water) was a control of maturation and effect of microinjection. Error bands represent ± SEM values. Different superscripts indicate significant differences (P<0.05). (B) Histogram showing percentages of parthenogenetic activated oocytes after 2 hours treatment with 5 mM SNAP, 10 mM CPTIO or 10 µM A23187 injected or non-injected 40 mM NO scavenger CPTIO or water. Error bands represent ±SEM values. Different superscripts indicate significant differences (P<0.05). Data are shown as mean percentage of activation of five independent experiments. (C) Western blot analysis. After 2 hours ocytes were taken off, homogenized in lysis buffer, and immunoblotted with antibodies against Xp42Mpk1 and p90Rsk. (D) Control oocyte in metaphase II arrested oocytes exhibits typical bipolar spindle with chromosomes and the first polar body (nuclear red/picro-indigo carmine staining). (E) Activation after A23187 treatment: typical pronucleus. (F) Oocyte activated by SNAP with pronucleus. (G) Oocyte after SNAP&CPTIO treatment in metaphase II. Scale bars represent 10 µm (D,G) and 40 µm (E,F).
Calcium limited medium (120 mM NaCl, 7.5 mM KCl, 500 µM MgSO4, 150 µM CaCl2, HEPES 22.5 µM, EDTA 400 µM, pH 7.4) and calcium free medium (96 mM NaCl, 2 mM KCl, 5 mM MgCl2, HEPES 5 mM, EGTA 500 µM, pH 7.4) were prepared 1 hour before using. Stock solution of Ca(2+)-ionophore A23187 (Boehringer Mannheim) (100 mM), NOR5 (200 mM) (Alexis Corp.) and BAPTA-AM (100 mM) (Sigma-Aldrich) were made in DMSO and stored at −20°C. NO donors SNAP (Alexis Corp.) and SNP (Sigma-Aldrich), and also nitric oxide scavenger - CPTIO (Alexis Corp.) were prepared fresh in appropriate mediums (1 hour before using).
The NO-scavenger CPTIO were micro-injected into immature oocytes, by the use of a positive displacement digital micropipette (Nichiryo) in ND96. After injection of NO-scavenger (15 nl of 40 mM stock solution), oocytes were allowed to recover for at least 1 hour and were stimulated by progesterone for overnight maturation.
Effects of 5 mM SNAP application on mature oocyte in ND96 (black triangles) or in ND96 with 50 µM BAPTA-AM (white squares) (A) or in presence of CPTIO (B). Typical results are depicted. Fluorescence was expressed in arbitrary units and background and auto-fluorescence were subtracted. SNAP superfusion, which started at time = 5 minutes, was responsible for a rise of intracellular calcium fluorescence. The latter was abolished by presence of CPTIO (C) Effect of calcium chelator on SNAP induced activation. Histogram showing percentages of activated oocytes after 2 hours treatment with 5 mM SNAP or 10 uM A23187 supplemented 50 µM or 100 µM BAPTA-AM. Oocyte in control group were treated for 2 hours in clear ND96. Error bands represent ±SEM values. Different superscripts indicate significant diferences (P<0.05). Data are shown as mean percentage of activation of minimally four independent experiments. (D) Percentages of parthenogenetic activated oocytes after 2 hours treatment with 5 mM SNAP in ND96 medium, calcium free medium (CaFree) and calcium limited medium (CaLim). Error bands represent ±SEM values. Different superscripts indicate significant differences (P<0.05). Data are shown as mean percentage of activation of minimally four independent experiments. (E) Western blot analysis. After 2 hours oocytes were taken off, homogenized in lysis buffer, and immunoblotted with antibodies against Xp42Mpk1 and p90Rsk. (F) Releasing of nitric oxide in calcium restricted mediums. Nitric oxide contents after SNAP treatment was determined in ND96, CaLim and CaFree mediums by colorimetric measurement of NO nitric oxide metabolites, nitrites and nitrates (NO3−/NO2). Each measurement was repeated three times. Different superscripts indicate significant differences (P<0.05).
Cytological Analysis and Cortical Granule Lectins Secretion Assay
For cytological analysis, oocytes were fixed overnight in Smiths fixative, dehydrated, and embedded in paraffin. Sections (7 µm thickness) were stained with nuclear red to detect nuclei and chromosomes and with picroindigo carmine that reveals cytoplasmic structures .
Cortical granule lectins secretion in response to SNAP was assayed using SDS-PAGE . For gel analysis (unless otherwise stated), individual oocytes were incubated in 15 µl of ND96 medium with SNAP at the concentration 5 mM. After 60 min, media were collected and added with one volume of Laemmli 2X buffer 4% beta-mercaptoethanol and incubated for 5 min at 95°C. Lectins were separated on 12.5% gels using molecular weight markers from Bio-Rad and either stained using SYPRO®Ruby.
Electrophoresis and Western-blotting
Eggs were lysed in homogenization buffer and centrifuged for 5 min at 10 000×g (4°C) to eliminate yolk platelets. Supernatants were added with one volume of Laemmli 2X buffer 4% beta-mercaptoethanol, heated at 100°C for 3 min and stored at −20°C until analysis. Proteins from oocytes were separated by SDS-PAGE (15–17%) and transferred onto a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech, United Kingdom). Blots were blocked with 5% low fat dry milk and incubated with specific antibody. p90Rsk was detected using the polyclonal rabbit antibody (p90Rsk1 C-21 sc231, Santa Cruz Biotechnology, 1/1000), Xp42MPK1 was detected using mouse monoclonal antibody (Erk2 D-2 sc1647, Santa Cruz Biotechnology, 1/500), Cyclin B was detected using mouse monoclonal antibody (sc-53239, Santa Cruz Biotechnology, 1/1250), anti-mos (C237, Santa Cruz Biotechnology, 1/5 000), anti-P-H3 antibodies (S10, Cell Signaling, 1/5 000) and P-Tyr15 Cdk1 using overnight incubation with mouse monoclonal antibody (tyr15 Cell, 1/10 000). Nitrocellulose membranes with bounded primary antibody were then incubated with appropriate secondary antibodies (1/2500). The signals were detected through chemiluminescent assay (ECL blotting detection kit, Amersham Pharmacia Biotech, United Kingdom).
Immunoprecipitation of cdk1 was performed on batches of 30 oocytes lysed in 300 µl of buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 0.05% SDS, 0.5% Triton X100, 5 mM MgCl2, 1 mg/ml bovine serum albumin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml benz-amidine, 1 mM PMSF, 1 mM sodium vanadate). After a centrifugation at 4°C for 15 min at 10,000 g, supernatants were incubated with anti-cdk1 (1∶200; Invitrogen) antibodies for 2 h at 4°C. Protein A-Sepharose beads (5 mg, Sigma) were added for 1 h at 4°C. Immune complexes were collected by centrifugation, rinsed three times, resuspended in Laemmli sample buffer, and subjected to a 10% SDS-PAGE. Immune complexes were analyzed by Western blotting using anti-cdk1 (A17, Santa Cruz Biotechnology, 1/10 000) and anti-cyclin B2 (X121.10, Santa Cruz Biotechnology, 1/5 000) antibodies and the advanced ECL detection system (Amersham Biosciences).
Prior imaging experiments, meiotic resumption was induced by application of 10 µM progesterone on stage VI Xenopus oocytes (see first section of Methods). For calcium imaging, oocytes were loaded with the calcium sensitive non-ratiometric fluorescent probe Fluo4 (acetoxy-methyl-ester derivative, Fluo4-AM, 25 µM; excitation performed at 494 nm and emission at 516 nm). The dyes were allowed at least 5 hours (Fluo4-AM) to equilibrate within the oocytes.
Fluorescence experiments were performed at room temperature (18–20°C) in ND96 solution with 5 mM SNAP. Some of these trials were conducted with BAPTA-AM, a well-known calcium chelator. Matured oocytes, at metaphase II stage, were loaded with 50 µM BATA-AM for 30 minutes , . Acquisitions were done (every minute) on the animal hemisphere of the oocytes using an epifluorescence scanning system fitted to a Nikon microscope (Paris, France). Images were collected with Nikon® software (NIS Elements AR 3.00) and analyzed using ImageJ® software (National Institutes of Health, Bethesda, MD). Fluorescence was expressed in arbitrary units and background and auto-fluorescences were subtracted. Data obtained out of these experiments were from multiple oocytes (at least 3 batches and 3 oocytes for each).
Measurement of Nitric Oxide Metabolites
Nitric oxide was determined by colorimetric measurement of NO nitric oxide metabolites, nitrites and nitrates (NO3−/NO2−). Nitrates were enzymatically converted by NO–reductase into nitrites. Nitrites were quantified using Griess reagents (p–aminobenzensulfonamide in 3.0 N HCl and N–(1–naftyl) ethylendiamin dichloride). The amount of nitrites was measured spectrophotometrically on microtitration plates using an ELISA plate reader Rainbow (wavelength 520 nm, Austria, SLT).
The number of experimental replicates is indicated in the figure legends. Data are presented as means ± S.E.M. The data were analysed by analysis of variance (Scheffés test) using statistical software (STATISTICA 8.0 StatSoft). A value of P<0.05 was considered as statistically significant.
NO Donors SNAP Induces the Morphological Events of Egg Activation
The effects of nitrosothiol derivative (SNAP) – NO donor on eggs from 19 different females are summarized in Table 1. Concentrations of SNAP were chosen according to concentrations previously used in porcine oocytes in a range from 0.1 to 5.0 mM , . No effects were observed with low concentrations of SNAP (1 mM and 2 mM) on the maintenance of the metaphase II arrest in treated eggs : matured oocytes still exhibited a white spot, where the spindle is anchored. White spot occurs during maturation when the nucleus, or germinal vesicle, moves toward the apex of cells, pushing aside the pigments. Pigment rearrangement were observed in 40% of 2.5 mM SNAP-treated eggs while 77.1% of eggs treated with 5 mM SNAP exhibited pigment rearrangement typical of those observed with control calcium ionophore A23187: the white spot disappeared while pigments were concentrated in the animal hemisphera, moving toward the apex of the cell (Fig. 1B vs 1C). Using Gundersen et al’s protocol , we assessed cortical granules lectins presence in 5 mM-treated eggs (Fig. 1D). SNAP induced release of cortical granule lectins in metaphase II-blocked eggs but had no effect on immature oocytes arrested in prophase I since no lectins were detected in the incubating medium (Fig. 1D). Thus, SNAP induces morphological changes related to those of cortical reaction, which are also observed for egg activation induced by calcium ionophore A23187.
Other NO donors were tested at similar concentrations (Table 2), including NOR5 and SNP, but were discarded because of (1) being respectively less potent to induce NO release (data not shown), (2) producing CN− ions production, which can have secondary effects (data not shown, see also , ), (3) the toxicity of DMSO vehicle and (4) being less cell-permeant than SNAP.
SNAP Induces MAPK Inactivation without Impairing MPF
To further characterize the effects of NO donor SNAP, western blotting analysis of meiotic key components involved in metaphase II block was performed (Fig. 2). We observed that the external signs of SNAP-induced egg activation were detected after longer time exposure in comparison to calcium ionophore (Fig. 2A) (delay 70.17+/−20.11 min). While metaphase II arrested eggs exhibited Xp42MPK1 and its downstream effector p90Rsk under their phosphorylated and active isoforms, SNAP exposure drives the inactivation of both proteins within 30 min after the addition of the NO donor (Fig. 2B). Noticeably, Mos was not degraded and remained at constant level in this condition, in contrast to A23187-treated eggs where it is degraded after 70 minutes (Fig. 2C). MPF activity was not impaired by SNAP treatment: Cyclin level remained stable, Cdk1 was not phosphorylated on tyrosine 15 and histone H3 was phosphorylated, which attested the activity of MPF (Fig. 2C). Moreover, in order to determine if both components of MPF were still in complex, immunoprecipitation were performed using antibodies against Cdk1. Such strategy revealed that both partners were still in a complex (Fig. 2D). Control oocytes treated by Calcium Ionophore A23187 exhibited a drop in MPF activity (Cyclin B degradation, absence of phospho-Histone H3).
SNAP Effects are Dependent upon NO Release
We investigated the effect of NO scavenger (CPTIO) microinjection on NO-induced eggs activation. To efficiently overcome the effects of NO-donor SNAP, CPTIO was added in ND96 prior to the stimulation of maturation by progesterone. NO scavenger did not impact maturation in these conditions (10 mM, Fig. 3A). When 5 mM SNAP were applied on CPTIO microinjected eggs, no morphological changes were observed (Fig. 3B) and both Xp42MPK1 and p90Rsk remained phosphorylated (Fig. 3C). Furthermore, classical histological analysis revealed that after injection by CPTIO, SNAP-treated eggs remained in metaphase II (Table 3; Fig. 3D–G). In SNAP-treated eggs, pronuclei were observed (11.4% at membrane and 34.3% pronuclei migrated in the subcortical area, Table 3) but no metaphase spindle has been detected, suggesting that the latter has been disorganized due to NO release effects. Then, while CPTIO did not block the effects of A23187 but suppresses the effects of SNAP, both morphological and biochemical changes observed in SNAP treated oocytes have to be attributed to NO release and not to non-specific effects of SNAP itself.
SNAP Effects Depend upon Calcium Increase
Finally we tested the hypothesis that the activation of Xenopus eggs via the NO-donor SNAP was mediated through calcium increase or mobilization. We observed a strong increase of intracellular calcium fluorescence induced by SNAP within the first 30 min of the treatment (Fig. 4A). This effect was specific towards NO release since CPTIO treatment completely abolished the increase observed in presence of SNAP (Fig. 4B). Treatment of oocytes with BAPTA-AM suppressed the effect of SNAP on intracellular calcium changes but BAPTA-AM alone failed to block SNAP induced cortical reaction and pigment rearrangement (Fig. 4C). We subsequently tested two different media: calcium free medium (CaFree) and calcium limited medium (CaLim), the latter being less drastic for calcium deprivation and known to prevent mechanical and spontaneous parthenogenetic activations. In our hands, both media impaired eggs activation induced by SNAP (57.2+/−5.9, 11.9+/−2.9 and 0+/−0, respectively for SNAP incubation in ND96, calcium free medium and calcium limited medium (Fig. 4D). Western blot analyses confirmed that p90Rsk and Xp42MPK1 remained phosphorylated and under an active state after 2 hours treatment in calcium free or calcium limited medium (Fig. 4E). Releasing of NO from SNAP was similar in all three tested mediums (Fig. 4F).
The role of NO-dependent signaling cascade in egg activation remains unclear and is subjected to intensive research. NO donors have been considered as potential tool to promote parthenogenetic activation in different modes, such as sea urchin  and porcine oocytes , while NO may be also involved in mechanisms of fertilization in sea urchin  or mouse oocytes . NO signaling molecules and NO levels have become issues in parthenogenetic activation and in vitro culture of oocytes in vertebrate’s models. Here we took advantages of the amphibian model (amenability to biochemical studies, one living cell imaging, ease of manipulation and culture independent towards follicular cells) to test the hypothesis that the NO-donor (SNAP) could induce parthenogenetic activation and exit from the metaphase II block in lower vertebrate eggs.
Several nitric oxide donors were tested: SNAP (S–nitroso-N-Acetyl-D,L-penicillamine), SNP (Sodium nitroprusside) and NOR5 (±)-2-((E)-4-Ethyl-3[(Z)-hydroxyimino]6-methyl-5-nitro-heptenyl)-3-pyridinecarboxamide). SNP and NOR5 were rapidly discarded, and SNAP was chosen due to its effects on Xenopus eggs and its ease of use in our context. NOR5 and SNP did not exert any effects on metaphase II block (data not shown); SNP is regarded as less potent than SNAP to induce NO release ( ; M. Jeseta, personal observations), has to be exposed to light for nitric oxide release  and finally may produce cyanide CN- ions following its breakdown , . The absence of effects of NOR5 at the concentrations used (2 mM) may be due to the fact that this compound is believed to be less permeant than SNAP. Its injection into Xenopus eggs was not considered because of NOR5 being solubilized in DMSO, which could exert itself deleterious effects on metaphase II block .
SNAP is a stable analogous of endogenous S-nitrosothiols, and NO is released from it by endogenous enzymes after the penetration of SNAP into the cell –. On a physiological note, endogenous NO may be produced by three different isoforms of NOS. The presence of eNOS and iNOS isoforms was observed in fully-grown rat , mouse , porcine ,  cattle oocytes . In Xenopus, only nNOS was detected . Similarly to porcine eggs, SNAP long time exposures were requested to observe external signs of parthenogenetic activation, as attested by pigment rearrangement . Eggs permeability to NO-donors or NO releasing rates may be determining and limiting factors in the slow dynamic of cortical reaction observed in these models. In our hands, SNAP induced cortical granules exocytosis, in contrast to porcine oocytes treated with NO-donor, which did not exhibit the exocytosis of cortical granules .
Because cortical reactions can be considered as deconnected from metaphase II exit per se, we checked for the activity and activation status of MPF and MAPK. MAPK and its downstream effector p90rsk were dephosphorylated within 30 minutes post-addition of SNAP whereas MPF remained under an active profile (tyrosine 15 unphosphorylated and maintenance of high levels of Cyclin B). Inhibition of MPF activity could have been driven in these conditions by the dissociation of Cdk1 from its regulatory subunit. The Cdk1-Cyclin B dissociation is sufficient to dramatically impair MPF activity , . Nevertheless, in our hands, SNAP exposure failed to promote the disassembly of the MPF, because both partners of the heterodimer, Cdk1 and Cyclcin B, were immunoprecipitated together. Finally, maintenance of MPF activity was firmly suggested by the phosphorylation of Histone H3 and the absence of Mos degradation. Indeed, Mos degradation is prevented by the activity of MPF, which phosphorylates Mos on its residue serine 3, thus protecting the protein from its degradation . Then, MAPK activity declines while MPF remains active in SNAP-treated eggs. Interestingly, the decrease of MAPK activity was correlated to the disorganization of spindle in SNAP-treated eggs. Pigments trails from pronucleus migration and pronuclei were observed in these conditions. Similarly, interphase transition has been reported in metaphase II-blocked porcine oocytes treated with MEK inhibitor U0126 , .
CPTIO was successfully used in different cells types, including gametes and embryos, to block NO-release –. It has been previously reported differences in mammalian oocytes regarding sensitivity on nitric oxide during maturation. High concentration of NO donors supress nuclear maturation in bovine  and porcine oocyte . However low doses of NO donor stimulate nuclear maturation in mouse  or cattle oocyte . On the oher hand NOS inhibitors block nuclear maturation in pig  or mouse oocyte . In our conditions, NO-scavenging did not appear to impair the progression of M-phase entry and maturation in Xenopus oocytes, because CPTIO-treated oocytes treated with progesterone resumed and completed meiosis like control ones (Fig. 3A). These results indicate that the role of NO for regulation in meiotic maturation is not crucial for Xenopus oocytes maturation, in contrast to mammalian oocytes. Nevertheless, microinjection of NO scavenger blocked the effects of SNAP on eggs, enabling us to discard the hypothesis that SNAP effects are non specific: MAPK inactivation, spindle disorganization and pronuclei formation induced by SNAP were indeed severely impaired by CPTIO.
From literature, calcium- and NO-dependent signaling pathways are clearly interconnected in oocytes and eggs with calcium playing a pivotal and main role , , . The NO-dependent signaling cascade is regulated by calcium. The important role of calcium for NO induced activation was also confirmed by our study, which demonstrated that NO-induced parthenogenetic activation is impaired in calcium limited or calcium free media. Production of NO in the cell depends on activation of NOS by Calmodulin, which is itself activated by calcium ions , . Mainly, NO influences the levels of intracellular calcium through the regulation of calcium ion channels and pumps, which modulate the influx and efflux of calcium between the cell and extracellular space . NO could also regulate the mobilization of calcium ions from their intracellular stores. Calcium from this source is released through either ryanodine (RYR) or inositol triphosphate receptors (IP3R) . Interestingly NO-dependent activation of pig oocytes depends only on mobilization of intracellular calcium stores from RYRs. The mobilization of endogenous calcium stores through IP3R is not necessary for SNAP parthenogenetic activation in porcine .
In Xenopus laevis oocytes the study of the elementary events of calcium signalling is facilitated by the lack of ER calcium-release channels (e.g., ryanodine receptors (RYRs) and cADP-ribose receptors) other than IP3Rs . In fact, RYR are poorly expressed and located near nucleus in Xenopus oocytes. Therefore, the role of RYR is rather considered as modest for endogenous calcium release mechanisms in contrast to pig oocytes , . Our results suggest that nitric oxide specifically induces parthenogenetic activation in Xenopus laevis eggs, through a calcium dependent mechanism, though the origin of the calcium changes in our context remains to be determined. Cell cycle control and progression, such as maturation and activation processes, imply different ionic modifications, including particularly calcium . Calcium sources could be differently mobilized and result in dramatic as well as discrete changes, at local levels. For example, it is well known that, in mature oocyte, IP3 receptors are clustering at the membrane of the endoplasmic reticulum , . This could be responsible for calcium variations in restricted areas – microdomains - where calcium concentration could rise but may be also difficult to detect and measure by oocyte imaging.
Inactivation of MAPK at metaphase II, while MPF is still active, offers an atypical playground to unravel the effects of NO on cell cycle regulation mechanisms at fertilization. In one hand, NO induced MAPK inactivation and cortical granule exocytosis through a calcium increase but on another hand, the increase in calcium failed to inactivate MPF. Why does not the calcium increase drive the inactivation of MPF? Nitrosylation has been reported in other cellular models to impair CaMKII activity , . Then, NO increase may lead to the inactivation of CaMKII and prevent the activation of APC, which is necessary for MPF inactivation and cyclin B degradation. Because calcium limited and calcium free media prevented the inactivation of MAPK induced by SNAP, a calcium-dependent event is involved in the inactivation of MAPK, and not nitrosylation itself. It was previously reported that nitric oxide induced activation of mammalian oocytes is stimulated by alternative signaling pathways, which are not involved in oocyte activation after treatment by traditional activating protocols (i.e. by calcium ionophore). Thus, mechanism of nitric oxide effect on matured oocytes remains to be determined. Further studies are also requested to clarify the role of nitric oxide in oocyte biology.
We thank Jean-Pierre Vilain for reading and fruitful discussions. The authors want to thank Elodie Richard of the BICeL-IFR147 Core Facility for access to instruments and technical advice.
Conceived and designed the experiments: JFB MJ MS JP. Performed the experiments: MJ MM KCM AM HT PM JFB RB. Analyzed the data: MJ JFB HT MM. Contributed reagents/materials/analysis tools: ALR RB MM. Wrote the paper: JFB MJ MM.
- 1. Lamas S, Marsden PA, Li GK, Tempst P, Michel T (1992) Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci USA 89: 6348–6352.S. LamasPA MarsdenGK LiP. TempstT. Michel1992Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.Proc Natl Acad Sci USA8963486352
- 2. Xie Q, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, et al. (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256: 225–228.Q. XieHJ ChoJ. CalaycayRA MumfordKM Swiderek1992Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.Science256225228
- 3. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43: 109–142.S. MoncadaRM PalmerEA Higgs1991Nitric oxide: physiology, pathophysiology and pharmacology.Pharmacol Rev43109142
- 4. Nathan C (1992) Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051–3064.C. Nathan1992Nitric oxide as a secretory product of mammalian cells.FASEB J630513064
- 5. Bian K, Murad F (2003) Nitric oxide (NO) – Biogeneration, regulation, and relevance to human diseases. Front Biosci 8: 264–278.K. BianF. Murad2003Nitric oxide (NO) – Biogeneration, regulation, and relevance to human diseases.Front Biosci8264278
- 6. Mehta JL, Chen CY, Kone BC, Mehta P, Turner P (1995) Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J Lab Clin Med 125: 370–377.JL MehtaCY ChenBC KoneP. MehtaP. Turner1995Identification of constitutive and inducible forms of nitric oxide synthase in human platelets.J Lab Clin Med125370377
- 7. Biswas S, Kabir SN, Pal AK (1998) The role of nitric oxide in the process of implantation in rats. J Reprod Fertil 114: 157–161.S. BiswasSN KabirAK Pal1998The role of nitric oxide in the process of implantation in rats.J Reprod Fertil114157161
- 8. Sengoku K, Takuma N, Horikawa M, Tsuchiya K, Komori H, et al. (2001) Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro. Mol Reprod Dev 58: 262–268.K. SengokuN. TakumaM. HorikawaK. TsuchiyaH. Komori2001Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro.Mol Reprod Dev58262268
- 9. Zini A, O’Bryan MK, Magid MS, Schlegel PN (1996) Immunohistochemical localization of endothelial nitric oxide synthase in human testis, epididymis, and vas deferens suggests a possible role for nitric oxide in spermatogenesis, sperm maturation, and programmed cell death. Biol Reprod 55: 935–941.A. ZiniMK O’BryanMS MagidPN Schlegel1996Immunohistochemical localization of endothelial nitric oxide synthase in human testis, epididymis, and vas deferens suggests a possible role for nitric oxide in spermatogenesis, sperm maturation, and programmed cell death.Biol Reprod55935941
- 10. Chmelikova E, Jeseta M, Sedmikova M, Petr J, Tumova L, et al. (2010) Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes. Zygote 29: 1–10.E. ChmelikovaM. JesetaM. SedmikovaJ. PetrL. Tumova2010Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes.Zygote29110
- 11. Bodart JF, Flament S, Vilain JP (2002) Minireview: Metaphase arrest in amphibian oocytes: Interaction between CSF and MPF sets the equilibrium. Mol Reprod Dev 61: 570–574.JF BodartS. FlamentJP Vilain2002Minireview: Metaphase arrest in amphibian oocytes: Interaction between CSF and MPF sets the equilibrium.Mol Reprod Dev61570574
- 12. Norbury C, Nurse P (1990) Controls of cell proliferation in yeast and animals. Ciba Found Symp 150: 168–173.C. NorburyP. Nurse1990Controls of cell proliferation in yeast and animals.Ciba Found Symp150168173
- 13. Fattaey A, Booher RN (1997) Myt1: a Wee1-type kinase that phosphorylates Cdc2 on residue Thr14. Prog Cell Cycle Res 3: 233–240.A. FattaeyRN Booher1997Myt1: a Wee1-type kinase that phosphorylates Cdc2 on residue Thr14.Prog Cell Cycle Res3233240
- 14. Rolfe M, Chiu M, Pagano M (1997) The ubiquitin-mediated proteolytic pathway as a therapeutic area. J Mol Med 75: 5–17.M. RolfeM. ChiuM. Pagano1997The ubiquitin-mediated proteolytic pathway as a therapeutic area.J Mol Med75517
- 15. Inoue D, Ohe M, Kanemori Y, Nobui T, Sagata N (2007) A direct link of the Mos-MAPK pathway to Erp1/Emi2 in meiotic arrest of Xenopus laevis eggs. Nature 446: 1100–1104.D. InoueM. OheY. KanemoriT. NobuiN. Sagata2007A direct link of the Mos-MAPK pathway to Erp1/Emi2 in meiotic arrest of Xenopus laevis eggs.Nature44611001104
- 16. Nishiyama T, Ohsumi K, Kishimoto T (2007) Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs. Nature 446: 1096–1099.T. NishiyamaK. OhsumiT. Kishimoto2007Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs.Nature44610961099
- 17. Wu JQ, Kornbluth S (2008) Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era. J Cell Sci 121: 3509–3514.JQ WuS. Kornbluth2008Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era.J Cell Sci12135093514
- 18. Knobil E, Neil , editors. Yanagimachi R. 1988. Mammalian fertilization. pp. 230–278.E. KnobilNeilYanagimachi R. 1988. Mammalian fertilization.230278editors.The Physiology of Reproduction.New York: Raven Press. New York: Raven Press.
- 19. Stricker SA (1999) Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev Biol 211: 157–176.SA Stricker1999Comparative biology of calcium signaling during fertilization and egg activation in animals.Dev Biol211157176
- 20. Ullah G, Jung P, Machaca K (2007) Modeling Ca2+ signaling differentiation during oocyte maturation. Cell Calcium 42: 556–564.G. UllahP. JungK. Machaca2007Modeling Ca2+ signaling differentiation during oocyte maturation.Cell Calcium42556564
- 21. Sun L, Hodeify R, Haun S, Charlesworth A, MacNicol AM, et al. (2008) Ca2+ homeostasis regulates Xenopus oocyte maturation. Biol Reprod 78: 726–735.L. SunR. HodeifyS. HaunA. CharlesworthAM MacNicol2008Ca2+ homeostasis regulates Xenopus oocyte maturation.Biol Reprod78726735
- 22. Bodart JF, Bechard D, Bertout M, Gannon J, Rousseau A, et al. (1999) Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos)proteolysis. Exp Cell Res 253: 413–421.JF BodartD. BechardM. BertoutJ. GannonA. Rousseau1999Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos)proteolysis.Exp Cell Res253413421
- 23. Flament S, Bodart JF, Bertout M, Browaeys E, Rousseau A, et al. (2000) Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs. Zygote 8: 3–14.S. FlamentJF BodartM. BertoutE. BrowaeysA. Rousseau2000Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs.Zygote8314
- 24. Bodart JF, Flament S, Vilain JP (2002) Metaphase arrest in amphibian oocytes: interaction between CSF and MPF sets the equilibrium. Mol Reprod Dev 61: 570–574.JF BodartS. FlamentJP Vilain2002Metaphase arrest in amphibian oocytes: interaction between CSF and MPF sets the equilibrium.Mol Reprod Dev61570574
- 25. Suzuki T, Suzuki E, Yoshida N, Kubo A, Li H, et al. (2010) Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase. Development 137: 3281–3291.T. SuzukiE. SuzukiN. YoshidaA. KuboH. Li2010Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase.Development13732813291
- 26. Busa WB, Ferguson JE, Joseph SK, Williamson JR, Nuccitelli R (1985) Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores. J Cell Biol 101: 677–682.WB BusaJE FergusonSK JosephJR WilliamsonR. Nuccitelli1985Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores.J Cell Biol101677682
- 27. Machaca K (2007) Ca2+ signaling differentiation during oocyte maturation. J Cell Physiol 213: 331–340.K. Machaca2007Ca2+ signaling differentiation during oocyte maturation.J Cell Physiol213331340
- 28. Busa WB, Nuccitelli R (1985) An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis. J Cell Biol 100: 1325–1329.WB BusaR. Nuccitelli1985An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis.J Cell Biol10013251329
- 29. Machaca K, Haun S (2000) Store-operated calcium entry inactivates at the germinal vesicle breakdown stage of Xenopus meiosis. J Biol Chem 275: 38710–38715.K. MachacaS. Haun2000Store-operated calcium entry inactivates at the germinal vesicle breakdown stage of Xenopus meiosis.J Biol Chem2753871038715
- 30. Marin M (2012) Calcium Signaling in Xenopus oocyte. In: Advances in Experimental Medicine and Biology. Vol 740, chapter 49, p. 875. In press. M. Marin2012Calcium Signaling in Xenopus oocyte.In: Advances in Experimental Medicine and Biology. Vol 740, chapter 49, p. 875. In press.
- 31. Steinhardt RA, Epel D (1974) Activation of sea-urchin eggs by a calcium ionophore. Proc Natl Acad Sci USA 71: 1915–1919.RA SteinhardtD. Epel1974Activation of sea-urchin eggs by a calcium ionophore.Proc Natl Acad Sci USA7119151919
- 32. El-Jouni W, Jang B, Haun S, Machaca K (2005) Calcium signaling differentiation during Xenopus oocyte maturation. Dev Biol 288: 514–525.W. El-JouniB. JangS. HaunK. Machaca2005Calcium signaling differentiation during Xenopus oocyte maturation.Dev Biol288514525
- 33. Kuo RC, Baxter GT, Thompson SH, Stricker SA, Patton C, et al. (2000) NO is necessary and sufficient for egg activation at fertilization. Nature 406: 633–636.RC KuoGT BaxterSH ThompsonSA StrickerC. Patton2000NO is necessary and sufficient for egg activation at fertilization.Nature406633636
- 34. Hyslop LA, Carroll M, Nixon VL, McDougall A, Jones KT (2001) Simultaneous measurement of intracellular nitric oxide and free calcium levels in chordate eggs demonstrates that nitric oxide has no role at fertilization. Dev Biol 234: 216–230.LA HyslopM. CarrollVL NixonA. McDougallKT Jones2001Simultaneous measurement of intracellular nitric oxide and free calcium levels in chordate eggs demonstrates that nitric oxide has no role at fertilization.Dev Biol234216230
- 35. Leckie C, Empson R, Becchetti A, Thomas J, Galione A, et al. (2003) The NO pathway acts late during the fertilization response in sea urchin eggs. J Biol Chem 278: 12247–12254.C. LeckieR. EmpsonA. BecchettiJ. ThomasA. Galione2003The NO pathway acts late during the fertilization response in sea urchin eggs.J Biol Chem2781224712254
- 36. Petr J, Rajmon R, Rozinek J, Sedmikova M, Jeseta M, et al. (2005a) Activation of pig oocytes using nitric oxide donors. Mol Reprod Dev 71: 115–122.J. PetrR. RajmonJ. RozinekM. SedmikovaM. Jeseta2005aActivation of pig oocytes using nitric oxide donors.Mol Reprod Dev71115122
- 37. Petr J, Rajmon R, Lanska V, Sedmikova M, Jilek F (2005b) Nitric oxide-dependent activation of pig oocytes: Role of calcium. Mol Cell Endocrinol 242: 16–22.J. PetrR. RajmonV. LanskaM. SedmikovaF. Jilek2005bNitric oxide-dependent activation of pig oocytes: Role of calcium.Mol Cell Endocrinol2421622
- 38. Amano T, Mori T, Matsumoto K, Iritani A, Watanabe T (2005) Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca2+ induced by inositol 1,4,5-triphosphate. Theriogenology 64: 261–274.T. AmanoT. MoriK. MatsumotoA. IritaniT. Watanabe2005Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca2+ induced by inositol 1,4,5-triphosphate.Theriogenology64261274
- 39. Bu S, Xia G, Tao Y, Lei L, Zhou B (2007) Dual effect of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro. Mol Cell Endocrinol 207: 21–30.S. BuG. XiaY. TaoL. LeiB. Zhou2007Dual effect of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro.Mol Cell Endocrinol2072130
- 40. Bodart JF, Beart FY, Sellier S, Duesbery NS, Flament S, et al. (2005) Differential roles of p39(Mos)-Xp42(Mpk1) cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes. Dev Biol 283: 373–383.JF BodartFY BeartS. SellierNS DuesberyS. Flament2005Differential roles of p39(Mos)-Xp42(Mpk1) cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes.Dev Biol283373383
- 41. Gundersen CB, Kohan SA, Chen Q, Iagnemma J, Umbach JA (2002) Activation of protein kinase Cη triggers cortical granule exocytosis in Xenopus oocytes. J Cell Sci 115: 1313–1320.CB GundersenSA KohanQ. ChenJ. IagnemmaJA Umbach2002Activation of protein kinase Cη triggers cortical granule exocytosis in Xenopus oocytes.J Cell Sci11513131320
- 42. Marin M, Sellier C, Paul-Antoine AF, Cailliau K, Browaeys-Poly E, et al. (2010) Calcium dynamics during physiological acidification in Xenopus oocyte. J Membr Biol 236: 233–245.M. MarinC. SellierAF Paul-AntoineK. CailliauE. Browaeys-Poly2010Calcium dynamics during physiological acidification in Xenopus oocyte.J Membr Biol236233245
- 43. Betts DH, Madan P (2008) Permanent embryo arrest: molecular and cellular concepts. Mol Hum Reprod 14: 445–453.DH BettsP. Madan2008Permanent embryo arrest: molecular and cellular concepts.Mol Hum Reprod14445453
- 44. Reyes R, Vázquez ML, Delgado NM (2004) Detection and bioimaging of nitric oxide in bovine oocytes and sperm cells. Arch Androl 50: 303–309.R. ReyesML VázquezNM Delgado2004Detection and bioimaging of nitric oxide in bovine oocytes and sperm cells.Arch Androl50303309
- 45. Yang JZ, Ajonuma LC, Rowlands DK, Tsang LL, Ho LS, et al. (2005) The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms. Cell Biol Int 29: 785–791.JZ YangLC AjonumaDK RowlandsLL TsangLS Ho2005The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms.Cell Biol Int29785791
- 46. Marks GS, McLaughlin BE, Jimmo SL, Poklewskakoziell M, Brien JF, et al. (1995) Time-dependent increase in nitric oxide formation concurrent with vasodilatation induced by sodium-nitropruside, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine but not by glyceryl trinitrate. Drug Metab Dispos 23: 1248–1252.GS MarksBE McLaughlinSL JimmoM. PoklewskakoziellJF Brien1995Time-dependent increase in nitric oxide formation concurrent with vasodilatation induced by sodium-nitropruside, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine but not by glyceryl trinitrate.Drug Metab Dispos2312481252
- 47. Butler AR, Glitwell C (1987) Recent chemical studies of sodium nitroprusside relevant to its hypotensive action. Chem Soc Rev 16: 361–380.AR ButlerC. Glitwell1987Recent chemical studies of sodium nitroprusside relevant to its hypotensive action.Chem Soc Rev16361380
- 48. Bates JN, Baker MT, Guerra R Jr, Harrison DG (1991) Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required. Biochem Pharmacol 42: 157–165.JN BatesMT BakerR. Guerra JrDG Harrison1991Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required.Biochem Pharmacol42157165
- 49. Yamamoto T, Bing RJ (2000) Nitric oxide donors. P Soc Exp Biol Med 22: 200–206.T. YamamotoRJ Bing2000Nitric oxide donors.P Soc Exp Biol Med22200206
- 50. Megson IL (2000) Nitric oxide donor drugs. Drug Future 25: 701–715.IL Megson2000Nitric oxide donor drugs.Drug Future25701715
- 51. Jablonka-Shariff A, Olson ML (1997) Hormonal regulation of nitric oxide synthases and their cell- specific expression during follicular development in the rat ovary. Endocrinology 138: 460–468.A. Jablonka-ShariffML Olson1997Hormonal regulation of nitric oxide synthases and their cell- specific expression during follicular development in the rat ovary.Endocrinology138460468
- 52. Nishikimi A, Matsukawa T, Hoshino K, Ikeda S, Kira Y, et al. (2001) Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice. Reproduction 122: 957–963.A. NishikimiT. MatsukawaK. HoshinoS. IkedaY. Kira2001Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice.Reproduction122957963
- 53. Hattori MA, Nishida N, Takesue K, Kato Y, Fujihara N (2000) FSH suppression of nitric oxide synthesis in porcine oocytes. J Mol Endocrinol 24: 65–73.MA HattoriN. NishidaK. TakesueY. KatoN. Fujihara2000FSH suppression of nitric oxide synthesis in porcine oocytes.J Mol Endocrinol246573
- 54. Chmelikova E, Sedmikova M, Petr J, Kott T, Lanska V, et al. (2009) Expression and localization of nitric oxide synthase isoforms during porcine oocyte growth and acquisition of meiotic competence. Czech J Anim Sci 54: 137–149.E. ChmelikovaM. SedmikovaJ. PetrT. KottV. Lanska2009Expression and localization of nitric oxide synthase isoforms during porcine oocyte growth and acquisition of meiotic competence.Czech J Anim Sci54137149
- 55. Pires PRL, Santos NP, Adona PR, Natori MM, Schwarz KRL, et al. (2009) Endothelial and inducible nitric oxide synthases in oocytes of cattle. Anim Reprod Sci 116: 233–243.PRL PiresNP SantosPR AdonaMM NatoriKRL Schwarz2009Endothelial and inducible nitric oxide synthases in oocytes of cattle.Anim Reprod Sci116233243
- 56. Peunova N, Scheinker V, Cline H, Enikolopov G (2001) Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain. J Neurosci 21: 8809–8818.N. PeunovaV. ScheinkerH. ClineG. Enikolopov2001Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain.J Neurosci2188098818
- 57. Lorca T, Fesquet D, Zindy F, Lebouffant F, Cerruti M, et al. (1991) An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway inamphibian eggs. Mol Cell Bio 11: 1171–1175.T. LorcaD. FesquetF. ZindyF. LebouffantM. Cerruti1991An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway inamphibian eggs.Mol Cell Bio1111711175
- 58. Chesnel F, Bazile F, Pascal A, Kubiak JZ (2006) Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos. Cell Cycle 5: 1687–1698.F. ChesnelF. BazileA. PascalJZ Kubiak2006Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos.Cell Cycle516871698
- 59. Doran SA, Tran CH, Eskicioglu C, Stachniak T, Ahn K-Ch, et al. (2003) Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells. Am J Physiol Regul Integr Comp Physiol 285: 348–355.SA DoranCH TranC. EskiciogluT. StachniakK-Ch Ahn2003Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells.Am J Physiol Regul Integr Comp Physiol285348355
- 60. Castro A, Peter M, Magnaghi-Jaulin L, Vigneron S, Galas S, et al. (2001) Cyclin B/cdc2 induces c-Mos stability by direct phosphorylation in Xenopus oocytes. Mol Biol Cell 2: 2660–2671.A. CastroM. PeterL. Magnaghi-JaulinS. VigneronS. Galas2001Cyclin B/cdc2 induces c-Mos stability by direct phosphorylation in Xenopus oocytes.Mol Biol Cell226602671
- 61. Fan HY, Tong C, Lian L, Li SW, Gao WX, et al. (2003) Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization. Biol Reprod 68: 968–977.HY FanC. TongL. LianSW LiWX Gao2003Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization.Biol Reprod68968977
- 62. Tatemoto H, Muto N (2001) Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes. Zygote 9: 15–23.H. TatemotoN. Muto2001Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes.Zygote91523
- 63. Stricker S (2011) Inhibition of germinal vesicle breakdown by antioxidants and the roles of signaling pathways related to nitric oxide and cGMP during meiotic resumption in oocytes of a marine worm. Reproduction 143: 261–270.S. Stricker2011Inhibition of germinal vesicle breakdown by antioxidants and the roles of signaling pathways related to nitric oxide and cGMP during meiotic resumption in oocytes of a marine worm.Reproduction143261270
- 64. Kumar S, Barthwal MK, Dikshit M (2010) Cdk2 nitrosylation and loss of mitochondrial potential mediate NO-dependent biphasic effect on HL-60 cell cycle. Free Radic Biol Med 48: 851–861.S. KumarMK BarthwalM. Dikshit2010Cdk2 nitrosylation and loss of mitochondrial potential mediate NO-dependent biphasic effect on HL-60 cell cycle.Free Radic Biol Med48851861
- 65. Viana KS, Caldas-Bussiere MC, Matta SG, Faes MR, de Carvalho CS, et al. (2007) Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes. Anim Reprod Sci 102: 217–227.KS VianaMC Caldas-BussiereSG MattaMR FaesCS de Carvalho2007Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes.Anim Reprod Sci102217227
- 66. Tichovska H, Petr J, Chmelikova E, Sedmikova M, Tumova L, et al. (2011) Nitric oxide and meiotic competence of porcine oocytes. Animal 5: 1398–1405.H. TichovskaJ. PetrE. ChmelikovaM. SedmikovaL. Tumova2011Nitric oxide and meiotic competence of porcine oocytes.Animal513981405
- 67. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21.MJ BerridgeP. LippMD Bootman2000The versatility and universality of calcium signalling.Nat Rev Mol Cell Biol11121
- 68. Clapham DE (1995) Calcium signaling. Cell 80: 259–268.DE Clapham1995Calcium signaling.Cell80259268
- 69. Erickson ES, Mooren OL, Moore-Nichols D, Dunn RC (2004) Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex. Biophys Chem 112: 1–7.ES EricksonOL MoorenD. Moore-NicholsRC Dunn2004Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex.Biophys Chem11217
- 70. Ajmat MT, Bonilla F, Zelarayán L, Bühler MI (2011) Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation. Zygote 19: 171–180.MT AjmatF. BonillaL. ZelarayánMI Bühler2011Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.Zygote19171180
- 71. Whitaker M (2006) Calcium microdomains and cell cycle control. Cell Calcium 40: 585–592.M. Whitaker2006Calcium microdomains and cell cycle control.Cell Calcium40585592
- 72. Berridge MJ (1996) Microdomains and elemental events in calcium signaling. Cell Calcium 20: 95–96.MJ Berridge1996Microdomains and elemental events in calcium signaling.Cell Calcium209596
- 73. Berridge MJ (2006) Calcium microdomains: organization and function. Cell Calcium 40: 405–412.MJ Berridge2006Calcium microdomains: organization and function.Cell Calcium40405412
- 74. Song T, Hatano N, Kambe T, Miyamoto Y, Ihara H, et al. (2008) Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II. Biochem J 412: 223–231.T. SongN. HatanoT. KambeY. MiyamotoH. Ihara2008Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II.Biochem J412223231
- 75. Takata T, Kimura J, Tsuchiya Y, Naito Y, Watanabe Y (2011) Calcium/calmodulin-dependent protein kinases as potential targets of nitric oxide. Nitric Oxide 25: 145–152.T. TakataJ. KimuraY. TsuchiyaY. NaitoY. Watanabe2011Calcium/calmodulin-dependent protein kinases as potential targets of nitric oxide.Nitric Oxide25145152