Effect of three different nitric oxide donors on parthenogenetic activation of <i>Xenopus laevis</i> matured oocytes.

(A–B)<p>Values with different superscripts within the same row are significantly different.</p>*<p>high concetration of NOR5 strongly increase rate of lytic oocytes.</p><p>Data are shown as mean percentage of activation of five independent experiments for each experimental group.</p

Effect of SNAP, A23187 and SNAP+CPTIO on spindle morphogenesis and pronuclear formation following progesterone-induced maturation.

<p>Effect of SNAP, A23187 and SNAP+CPTIO on spindle morphogenesis and pronuclear formation following progesterone-induced maturation.</p

SNAP induced parthenogenetic activation is calcium-dependent.

<p>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 Xp42^Mpk1 and p90^Rsk. (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).</p

NO scavenger CPTIO does not impair maturation by, progesterone but suppresses SNAP effects on metaphase-II arrested eggs.

<p>(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 Xp42^Mpk1 and p90^Rsk. (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).</p

Kinetic of SNAP induced activation.

<p>(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, Xp42^Mpk1 and p90^Rsk. (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.</p

Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

<div><p>Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H₂S) in the process of porcine oocyte aging. H₂S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H₂S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H₂S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H₂S from a donor (Na₂S.9H₂O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H₂S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H₂S on MPF and MAPK activities were detected and the intracellular mechanism underlying H₂S activity remains unclear, our study clearly demonstrates the role of H₂S in the regulation of porcine oocyte aging.</p></div

Nitric oxide donor SNAP induces cortical reaction typical of parthenogenetic activation in <i>Xenopus laevis</i> matured oocytes.

<p>(A) Typical morphologies of <i>Xenopus laevis</i> 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 <i>Xenopus</i> 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.</p

Parthenogenetic activation of oocytes aged under the effect of the H₂S donor.

<p>At Oocytes were cultivated 48 hours to the metaphase II and then divided into 4 groups (see table). Control group (MII) was parthenogenetically activated immediately (without any exposure to prolonged cultivation). Other groups were exposed to prolonged cultivation (aging) for 24 hours in modified M199 medium supplemented with a H2S donor (Na₂S.9H₂O; 0μM, 150μM, and 300μM) and then parthenogenetically activated with calcium ionophore (25μM, 5 min) combined with 6-dimethyl aminopurine (2mM, 2 h). Subsequently, oocytes were cultured in NCSU 23 medium for the following 24 hours.</p><p>^a,b,c Statistically significant differences in the ratio of activated oocytes between individual treatments (in columns) are indicated with different superscripts (P<0.05).</p><p>Parthenogenetic activation of oocytes aged under the effect of the H₂S donor.</p

Reversion of the effects of CBS, CSE and MPST inhibitors using a H₂S donor.

<p>Oocytes were cultivated to metaphase II and then exposed to prolonged cultivation (24 hours) in a modified M199 medium supplemented with a H₂S donor (Na₂S.9H₂O; 300 μM) and the following individual inhibitors: oxamic acid (1mM, OA), beta-kyano-L-alanine (1mM, KA), and alpha-ketoglutaric acid disodium salt dihydrate (5mM, KGA). <i>Na₂S (300 μM, Na₂S.9H₂O); KGA—alpha-ketoglutaric acid disodium salt dihydrate (5 mM); KA – beta-kyano-L-alanine (1mM); OA—oxamic acid (1 mM); MII—intact oocytes (oocytes at metaphase II, anaphase II or telophase II), A—activated oocytes (oocytes with pronuclei or embryos), F—fragmented oocytes, L—lysed oocytes; Different letters and numbers indicate significant differences between different treatments and hours of aging (P<0.05). A,B – statistically significant differences in portion of MII stage oocytes between individual treatments. a,b,c,d – statistically significant differences in portion of activated oocytes between individual treatments. 1,2,3 – statistically significant differences in portion of fragmented oocytes between individual treatments</i>.</p

Effect of H₂S donor on MPF and MAPK activity. 6A

<p>Histone H1 kinase assay was carried out to determine the activity of MPF by measurement of MPF capacity to phosphorylate its substrate (histone H1). <b>6B</b>: MBP kinase assay was carried out to determine the activity of MAPK by measurement of MAPK capacity to phosphorylate its substrate (MBP – Myelin basic protein). MPF and MAPK activities were determined in the MII oocytes (C – control, white column), the oocytes aged 12h and 24h in modified M199 medium, the oocytes aged 12h and 24h in modified M199 medium supplemented with a H₂S donor (Na₂S, black column), and the oocytes aged 12h and 24h in modified M199 medium supplemented with triple combination of inhibitors (3Ki, grey column). The results are presented as a ratio relative to the group of oocytes at metaphase II. <i>(GV – germinal vesicle stage; MII – oocytes at metaphase II; A12–12 hours of aging; A24–24 hours of aging; C – control, white column; Na₂S—Na₂S.9H₂O, 300 μM, black column; 3Ki - 1mM oxamic acid + 1mM beta-kyano-L-alanine + 5mM alpha-ketoglutaric acid disodium salt dihydrate). ^a,b, Statistically significant differences in activity (MPF or MAPK) between individual treatments at the same time are indicated with different superscripts (P<0.05)</i>.</p