Hydrogen Sulfide Impairs Meiosis Resumption in Xenopuslaevis Oocytes

Abstract

The role of hydrogen sulfide (H₂S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H₂S metabolism, cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H₂S. On another hand, a dose-dependent inhibition was obtained using the H₂S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H₂S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H₂S signaling in the timing of amphibian oocytes meiosis resumption.

Keywords: Xenopus laevis; cell cycle; hydrogen sulfide; meiosis; oocyte.

Figure 1

NaHS inhibits meiosis resumption induced by progesterone in a dose-dependent manner. Oocytes were preincubated in NaHS at different concentrations before progesterone addition (4 μg/mL). (A) Percentage of oocytes with a white spot 24 h after addition of progesterone. Statistical significance between control and NaHS conditions was accepted for * p < 0.05 and *** p < 0.001. Microphotographic of whole oocytes with (WS) or without (NWS) white spots and of bisected oocytes along the animal/vegetative axis after heating 15 min at 100 °C (WS-h/NWS-h) are shown. (B) Oocytes exhibiting white spots were scored every hour (0–10 h and 24 h–30 h). (C) Western blot of p90^Rsk and Erk2 (on modified polyacrylamide gels, the phosphorylated forms have a slower mobility shift), phospho-Tyr15-Cdk1 and phospho-ser10-H3. Some oocytes were maintained in culture medium without treatment (prophase I, PI) or treated by progesterone alone (metaphase II, MII). Oocytes were sorted into two groups: without (NWS) or with a white spot (WS). Experiments were performed on 20 oocytes per condition and three independent females.

Figure 2

Inhibitors of H₂S-releasing enzymes accelerate meiosis resumption. (A) Western blot detection of endogenous enzymes of the H₂S metabolism, CBS, CSE, and MPST, in untreated oocytes (PI) and oocytes treated by progesterone (4 μg/mL) (MII). Total phosphorylated and unphosphorylated forms of Erk2 serves as loading controls. (B) Oocytes were pre-incubated or not with a cocktail of H₂S-releasing enzymes inhibitors AOAA (aminooxyacetic acid-10 mM), PAG (dl-propargylglycine-10 μM), and KGA (ketoglutaric acid-10 μM) before progesterone (4 μg/mL) stimulation (Pg + AOAA + PAG + KGA) and their white spots were recorded every hour for 24 h. (C) GVBD₅₀ corresponded to the time required to obtain 50% of mature oocytes. GVBD₅₀ were normalized with the GVBD₅₀ of the oocytes treated with progesterone only. Twenty oocytes were used in each condition from 3 independent females. Statistical significance was accepted for ** p < 0.01.

Figure 2

Inhibitors of H₂S-releasing enzymes accelerate meiosis resumption. (A) Western blot detection of endogenous enzymes of the H₂S metabolism, CBS, CSE, and MPST, in untreated oocytes (PI) and oocytes treated by progesterone (4 μg/mL) (MII). Total phosphorylated and unphosphorylated forms of Erk2 serves as loading controls. (B) Oocytes were pre-incubated or not with a cocktail of H₂S-releasing enzymes inhibitors AOAA (aminooxyacetic acid-10 mM), PAG (dl-propargylglycine-10 μM), and KGA (ketoglutaric acid-10 μM) before progesterone (4 μg/mL) stimulation (Pg + AOAA + PAG + KGA) and their white spots were recorded every hour for 24 h. (C) GVBD₅₀ corresponded to the time required to obtain 50% of mature oocytes. GVBD₅₀ were normalized with the GVBD₅₀ of the oocytes treated with progesterone only. Twenty oocytes were used in each condition from 3 independent females. Statistical significance was accepted for ** p < 0.01.

Figure 3

H₂S production in Xenopus oocytes. (A) Endogenous H₂S measurement was performed in prophase 1 blocked oocytes (PI) and oocytes 3 or 24 h after progesterone (4 μg/mL) (Pg 3 h or 24 h) addition without or with a 1 h pre-incubation with three inhibitors of H₂S metabolism (AOAA + PAG + KGA). (B) Exogenous production of H₂S was detected in oocytes after a pre-incubation of 1 h with a NaHS donor at 100 μM, 500 μM, 1 mM, and 5 mM. For each condition, four independent experiments were performed with n = 40. Statistical significance was accepted for ** p < 0.01 and *** p < 0.001.

Figure 4

NaHS prevents protein synthesis. (A) Western blot of Myt-Myc and Shb-Myc tagged proteins, and Erk2 from immature oocytes (PI) or oocytes micro-injected with mRNA coding for a Myc tagged proteins after pre-incubation or not for 15 min in NaHS at different concentrations. (B) Western blot of endogenous Mos and Erk2 from ten pooled oocytes 15 h after NaHS treatments and progesterone induction (4 μg/mL). Relative intensity of Myc and Mos bands were normalized with the intensity of their respective loading control bands of Erk2. Myc, Mos, and Erk2 band intensity were estimated using Image J software.

Figure 5

High concentrations of NaHS inhibit meiosis resumption induced by micro-injection of mature oocyte cytoplasm. Immature oocytes were pre-incubated (or not) in NaHS at different concentrations for 1 h before microinjection of mature cytoplasms. Some oocytes were maintained in culture medium without treatment (PI). (A) Percentage of oocytes exhibiting a white spot 7 h after micro-injection of mature oocyte cytoplasm. Statistical significance was accepted for *** p < 0.001. N = 3; n = 15 (B) Western blot analysis of p90^Rsk, Erk2, and P-Tyr15-Cdk1 for three oocytes/NaHS conditions.

Figure 6

NaHS does not dissociate the pre-MPF complexes nor impact MPF activity. (A) Immunoblots of Cyclin B2, P-Tyr15-Cdk1, and total Cdk1 after Cdk1 immunoprecipitations. Protein extracts from immature oocytes (PI) were treated or not with NaHS at different concentrations (500 µM, 1 mM, and 5 mM) for 24 h at 4 °C. The protein extracts were incubated with Cdk1 antibody overnight before they were precipitated with Protein A-sepharose beads for 1 h at 4 °C. Input and output lines correspond to proteins extracts without the Cdk1 antibody but before and after protein A-sepharose bead incubation, respectively. MII corresponds to protein extracts from mature oocytes. (B) Relative MPF activity was examined by histone H1 kinase assay after exposure of protein extracts from mature oocytes (MII) to NaHS at different concentrations (100 µM, 500 µM, 1 mM, and 5 mM) or heated for 30 min at 100 °C. MPF activities correspond to the quantification values of p-histone H1 on H1 bands obtained by autoradiography on 3 independent experiments. Statistical significance was accepted for *** p < 0.001. N = 3; n = 15.

Figure 6

NaHS does not dissociate the pre-MPF complexes nor impact MPF activity. (A) Immunoblots of Cyclin B2, P-Tyr15-Cdk1, and total Cdk1 after Cdk1 immunoprecipitations. Protein extracts from immature oocytes (PI) were treated or not with NaHS at different concentrations (500 µM, 1 mM, and 5 mM) for 24 h at 4 °C. The protein extracts were incubated with Cdk1 antibody overnight before they were precipitated with Protein A-sepharose beads for 1 h at 4 °C. Input and output lines correspond to proteins extracts without the Cdk1 antibody but before and after protein A-sepharose bead incubation, respectively. MII corresponds to protein extracts from mature oocytes. (B) Relative MPF activity was examined by histone H1 kinase assay after exposure of protein extracts from mature oocytes (MII) to NaHS at different concentrations (100 µM, 500 µM, 1 mM, and 5 mM) or heated for 30 min at 100 °C. MPF activities correspond to the quantification values of p-histone H1 on H1 bands obtained by autoradiography on 3 independent experiments. Statistical significance was accepted for *** p < 0.001. N = 3; n = 15.

Figure 7

NaHS impairs human Cdc25C activity in vitro. The reactions were performed using 1 µg of human recombinant Cdc25C pre-incubated or not with NaHS (500 μM, 1 mM, or 5 mM). Results are shown as relative ratios to control without NaHS. Statistical significance was accepted for *** p < 0.001. Three independent experiments were performed.

Figure 8

NaHS effects on meiosis resumption are reversed by ROS scavengers. Percentage of oocytes with a white spot 24 h after addition of progesterone (4 μg/mL). Oocytes were pre-incubated (or not) in the presence of NaHS at different concentrations and rescued with supplementation by SOD (150 units) and catalase (80 units). Statistical significance was accepted for * p < 0.05 and *** p < 0.001. N = 3; n = 15.

Figure 9

NaHS has no effect on apoptosis in Xenopus oocytes. (A) Survival rates of mature (WS) oocytes in presence or absence (control) of NaHS at the concentration of 5 mM. The number of dead oocytes was determined every hour. Microphotographs of healthy (WS) or dead (Dead) oocytes are shown. (B) Western blot of p90^Rsk, Erk2, cyclin B2, and cytochrome C. Mature oocytes were incubated (or not) in NaHS at 5 mM for 8, 24, 48, 56, and 72 h. Some oocytes were maintained in culture medium without treatment (PI) or in progesterone (4 μg/mL) alone (MII).