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A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Pandey AN, Chaube SK - Biores Open Access (2014)

Bottom Line: The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly.These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2.The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF.

View Article: PubMed Central - PubMed

Affiliation: Cell Physiology Laboratory, Department of Zoology, Banaras Hindu University , Varanasi, India .

ABSTRACT
Hydrogen peroxide (H2O2) acts as a signaling molecule and modulates various aspects of cell functions in a wide variety of cells including mammalian germ cells. We examined whether a decreased level of intra-oocyte cyclic 3',5'-adenosine monophosphate (cAMP) leads to accumulation of H2O2, and if so, whether a moderate increase of H2O2 inactivates maturation promoting factor (MPF) during spontaneous resumption of meiosis in rat oocytes cultured in vitro. Removal of cumulus cells and culture of denuded oocytes in vitro significantly decreased oocyte cAMP level and led to spontaneous meiotic resumption from diplotene arrest. The reduced oocyte cAMP level was associated with an increased oocyte H2O2 level and reduced catalase activity. Exogenous supplementation of H2O2 induced meiotic resumption from diplotene arrest in a concentration- and time-dependent manner in oocytes treated with 0.1 mM of 3-isobutyl-1-methylxanthine, while dibutyryl-cAMP and 3-t-butyl-4-hydroxyanisole inhibited the stimulatory effect of exogenous H2O2. The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly. These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2. The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF. The MPF inactivation triggered spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

No MeSH data available.


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Representative photographs showing immunofluorescence intensity of cyclin B1 level in oocytes. Reduction in the levels of cyclin B1 (arrow) (B) are associated with spontaneous resumption of meiosis from diplotene arrest as compared to diplotene stage (A). (Bar=20 μM). The CTCF analysis of three independent oocytes further supports above observations (C). Data are mean±SEM of three independent experiments and analyzed by Student's t-test. *Significant (p<0.001) difference as compared to diplotene-arrested oocytes.
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f9: Representative photographs showing immunofluorescence intensity of cyclin B1 level in oocytes. Reduction in the levels of cyclin B1 (arrow) (B) are associated with spontaneous resumption of meiosis from diplotene arrest as compared to diplotene stage (A). (Bar=20 μM). The CTCF analysis of three independent oocytes further supports above observations (C). Data are mean±SEM of three independent experiments and analyzed by Student's t-test. *Significant (p<0.001) difference as compared to diplotene-arrested oocytes.

Mentions: As shown in Figure 6, a significant increase in the level of Thr-14/Tyr-15 phosphorylated CDK1 was observed in oocytes that underwent spontaneous resumption of meiosis after 3 h of in vitro culture (Fig. 6A) as compared to diplotene-arrested oocytes (Fig. 6B). The CTCF analysis further supports these observations (Fig. 6C). As shown in Figure 7, a significant reduction in the level of Thr-161 phosphorylated CDK1 was observed in oocytes that underwent spontaneous resumption of meiosis after 3 h of in vitro culture (Fig. 7A) as compared to diplotene-arrested oocytes (Fig. 7B). The CTCF analysis further strengthens our observations (Fig. 7C). However, the total phosphorylation status of CDK1 remained unchanged during diplotene arrest (Fig. 8A) as well as during meiotic resumption from diplotene arrest (Fig. 8B). The CTCF analysis using Image J software further confirmed the already reported observations (Fig. 8C). Further, cyclin B1 level was significantly reduced in oocytes that underwent meiotic resumption (Fig. 9B) as compared to diplotene-arrested oocytes (Fig. 9A). These observations were further supported by the CTCF analysis (Fig. 9C).


A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Pandey AN, Chaube SK - Biores Open Access (2014)

Representative photographs showing immunofluorescence intensity of cyclin B1 level in oocytes. Reduction in the levels of cyclin B1 (arrow) (B) are associated with spontaneous resumption of meiosis from diplotene arrest as compared to diplotene stage (A). (Bar=20 μM). The CTCF analysis of three independent oocytes further supports above observations (C). Data are mean±SEM of three independent experiments and analyzed by Student's t-test. *Significant (p<0.001) difference as compared to diplotene-arrested oocytes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4120648&req=5

f9: Representative photographs showing immunofluorescence intensity of cyclin B1 level in oocytes. Reduction in the levels of cyclin B1 (arrow) (B) are associated with spontaneous resumption of meiosis from diplotene arrest as compared to diplotene stage (A). (Bar=20 μM). The CTCF analysis of three independent oocytes further supports above observations (C). Data are mean±SEM of three independent experiments and analyzed by Student's t-test. *Significant (p<0.001) difference as compared to diplotene-arrested oocytes.
Mentions: As shown in Figure 6, a significant increase in the level of Thr-14/Tyr-15 phosphorylated CDK1 was observed in oocytes that underwent spontaneous resumption of meiosis after 3 h of in vitro culture (Fig. 6A) as compared to diplotene-arrested oocytes (Fig. 6B). The CTCF analysis further supports these observations (Fig. 6C). As shown in Figure 7, a significant reduction in the level of Thr-161 phosphorylated CDK1 was observed in oocytes that underwent spontaneous resumption of meiosis after 3 h of in vitro culture (Fig. 7A) as compared to diplotene-arrested oocytes (Fig. 7B). The CTCF analysis further strengthens our observations (Fig. 7C). However, the total phosphorylation status of CDK1 remained unchanged during diplotene arrest (Fig. 8A) as well as during meiotic resumption from diplotene arrest (Fig. 8B). The CTCF analysis using Image J software further confirmed the already reported observations (Fig. 8C). Further, cyclin B1 level was significantly reduced in oocytes that underwent meiotic resumption (Fig. 9B) as compared to diplotene-arrested oocytes (Fig. 9A). These observations were further supported by the CTCF analysis (Fig. 9C).

Bottom Line: The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly.These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2.The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF.

View Article: PubMed Central - PubMed

Affiliation: Cell Physiology Laboratory, Department of Zoology, Banaras Hindu University , Varanasi, India .

ABSTRACT
Hydrogen peroxide (H2O2) acts as a signaling molecule and modulates various aspects of cell functions in a wide variety of cells including mammalian germ cells. We examined whether a decreased level of intra-oocyte cyclic 3',5'-adenosine monophosphate (cAMP) leads to accumulation of H2O2, and if so, whether a moderate increase of H2O2 inactivates maturation promoting factor (MPF) during spontaneous resumption of meiosis in rat oocytes cultured in vitro. Removal of cumulus cells and culture of denuded oocytes in vitro significantly decreased oocyte cAMP level and led to spontaneous meiotic resumption from diplotene arrest. The reduced oocyte cAMP level was associated with an increased oocyte H2O2 level and reduced catalase activity. Exogenous supplementation of H2O2 induced meiotic resumption from diplotene arrest in a concentration- and time-dependent manner in oocytes treated with 0.1 mM of 3-isobutyl-1-methylxanthine, while dibutyryl-cAMP and 3-t-butyl-4-hydroxyanisole inhibited the stimulatory effect of exogenous H2O2. The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly. These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2. The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF. The MPF inactivation triggered spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

No MeSH data available.


Related in: MedlinePlus