Jeseta M et al. (2012), Nitric oxide-donor SNAP induces Xenopus eggs ac...

XB-ART-45913

PLoS One 2012 Jan 01;77:e41509. doi: 10.1371/journal.pone.0041509.

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Nitric oxide-donor SNAP induces Xenopus eggs activation.

Jeseta M , Marin M , Tichovska H , Melicharova P , Cailliau-Maggio K , Martoriati A , Lescuyer-Rousseau A , Beaujois R , Petr J , Sedmikova M , Bodart JF .

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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.

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Species referenced: Xenopus laevis
Genes referenced: adm cdk1 mapk1 mos rps6ka1

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	Figure 1. Nitric oxide donor SNAP induces cortical reaction typical of parthenogenetic activation in Xenopus laevis matured oocytes. (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 . (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 A23187. After 1 hour the fluid surrounding oocytes (15 ) was collected for analysis by SDS-PAGE and SYPRO Ruby staining. Molecular weight standards are indicated in kDa.
	Figure 2. Kinetic of SNAP induced activation. (A) Kinetic of parthenogenetic activation after SNAP treatment. Matured oocytes were cultured with 5 mM SNAP or 10 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.

	Figure 4. SNAP induced parthenogenetic activation is calcium-dependent.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).

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Ajmat, Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation. 2011, Pubmed