DMBA acts on cumulus cells to desynchronize nuclear and cytoplasmic maturation of pig oocytes

Song, Zhi-Qiang; Li, Xuan; Wang, Yankui; Du, Zhiqiang; Yang, Caixia

doi:10.1038/s41598-017-01870-6

Cited by 25 publications

(19 citation statements)

References 44 publications

(61 reference statements)

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“…Although, the fluorescence-based DCFH-DA staining is the widely used method for detecting intracellular ROS in oocytes and embryos (Bae, 2015;Mihalas et al, 2017;Song et al, 2017;Yang et al, 2018), several limitations are associated with the method (Choi et al, 2012;Kalyanaraman et al, 2012;Roesslein et al, 2013). Therefore, in this study, we developed an efficient and easy-to-perform alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos.…”

Section: Discussionmentioning

confidence: 99%

“…The end products are detected based on the fluorescence or color they produce (Gosalvez et al, 2017). The most commonly used method for detecting intracellular ROS in oocytes and embryos is the 2, 7 -dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay (Bae, 2015;Mihalas et al, 2017;Song et al, 2017;Yang et al, 2018). Oxidation of DCFH-DA by ROS results in the formation of a fluorescent product DCF that can be detected by fluorescence microscopy (Kalyanaraman et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos

Javvaji

Dhali

Francis

et al. 2020

Front. Cell Dev. Biol.

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Assessment of intracellular reactive oxygen species (ROS) is important for evaluating the developmental ability of cumulus-oocyte complexes (COC) and embryos. Although, fluorescence-based 2 ,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method is used widely for detecting intracellular ROS in COC and embryos, it is associated with several limitations. This study aimed to develop an alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos based on nitroblue tetrazolium (NBT) staining and bright-field microscopy. Nitroblue tetrazolium reacts with ROS and forms formazan precipitate that can be detected as dark purple/blue spots under bright-field microscope. Ovine COC were matured in vitro without (control) or with the supplementation of Interleukin-7 (IL-7; for stimulating intracellular ROS), Tempol (superoxide scavenger) or combination of IL-7 and Tempol. The matured COC were stained with NBT and the formation of intracellular formazan precipitates was assessed. Additionally, the matured COC were stained with DCFH-DA to compare the level of intracellular ROS. Further, ovine embryos (8-cell, morula, and degenerating) were generated in vitro and stained with NBT for assessing intracellular ROS. The level of intracellular ROS was expressed as the proportion (%) of the NBT stained area of oocytes, compact cumulus cell masses or embryos. The proportions of NBT stained area in the matured oocytes and cumulus cells was found significantly lesser in the control as compared to the IL-7 (1 and 5 ng/ml) treated groups. A similar trend in the intracellular ROS level was also observed in the matured COC, when assessed based on the DCFH-DA staining. Following the treatment with Tempol (100 mM), negligible NBT stained area in oocytes and cumulus cells was observed. The NBT staining patterns of the oocytes and cumulus cells following the combined

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos

Javvaji

Dhali

Francis

et al. 2020

Front. Cell Dev. Biol.

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“…Quantitative PCR was conducted in a 10 μl system including 5 μl fast-start universal SYBR green master mix, 0.3 μl primers, 1 μlcDNA, 3.7 μl RNase-free water) (Roche Molecular Systems) on the 7500 real-time PCR detection system (Applied Biosystems, Carlsbad, CA, USA), using the following thermal cycling parameters: 95 °C for 10 min and 40 cycles of 95 °C for 15 s, 60 °C for 60 s. Ywhag and β-actin were used as internal controls respectively for oocyte and blastocyst samples. Relative mRNA level was calculated using the 2 −ΔΔCt method 52 . The primers were designed using Primer5 software (Supplementary Table S2 ).…”

Section: Methodsmentioning

confidence: 99%

Ascorbic acid induces global epigenetic reprogramming to promote meiotic maturation and developmental competence of porcine oocytes

Liu

et al. 2018

Sci Rep

Self Cite

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L-ascorbic acid (Vitamin C) can enhance the meiotic maturation and developmental competence of porcine oocytes, but the underlying molecular mechanism remains obscure. Here we show the role of ascorbic acid in regulating epigenetic status of both nucleic acids and chromatin to promote oocyte maturation and development in pigs. Supplementation of 250 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P) during in vitro maturation significantly enhanced the nuclear maturation (as indicated by higher rate of first polar body extrusion and increased Bmp15 mRNA level), reduced level of reactive oxygen species, and promoted developmental potency (higher cleavage and blastocyst rates of parthenotes, and decreased Bax and Caspase3 mRNA levels in blastocysts) of pig oocytes. AA2P treatment caused methylation erasure in mature oocytes on nucleic acids (5-methylcytosine (5 mC) and N6-methyladenosine (m6A)) and histones (Histone H3 trimethylations at lysines 27, H3K27me3), but establishment of histone H3 trimethylations at lysines 4 (H3K4me3) and 36 (H3K36me3). During the global methylation reprogramming process, levels of TET2 (mRNA and protein) and Dnmt3b (mRNA) were significantly elevated, but simultaneously DNMT3A (mRNA and protein), and also Hif-1α, Hif-2α, Tet3, Mettl14, Kdm5b and Eed (mRNA) were significantly inhibited. Our findings support that ascorbic acid can reprogram the methylation status of not only DNA and histone, but also RNA, to improve pig oocyte maturation and developmental competence.

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“…Our results suggest that excessive NaF exposure increases the incidence of early apoptosis. Because both DNA damage and early apoptosis affect oocyte meiotic maturation 65,66 , these might be the mechanisms through which excessive NaF exposure affects porcine oocyte meiotic maturation and blocks further development.…”

Section: Discussionmentioning

confidence: 99%

157 Sodium Fluoride Exposure Exerts Toxic Effects on Porcine Oocyte Maturation

Nie

Liang

Cui

2018

Reprod. Fertil. Dev.

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Excessive long-term fluoride intake is associated with several health problems, including infertility. However, limited information is available on the toxic effects of fluoride exposure on the female reproductive system, especially oocyte maturation. In this study, we investigated the toxic effect of sodium fluoride (NaF) exposure on porcine oocyte maturation and its possible underlying mechanisms. The level of reactive oxygen species, glutathione (GSH), mitochondrial membrane potential, apoptosis, and DNA damage and cathepsin B in the blastocysts were observed by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen, Carlsbad, CA, USA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, anti-γH2A.X (Ser139) and Magic Red cathepsin B assay kit, respectively. Data obtained from the experimental groups were compared using Student’s t-test. Our results showed that treatment with 60, 100, and 150 μg mL−1 NaF significantly decreased the degree of cumulus cell expansion (CEI: 2.64 ± 0.27, 1.32 ± 0.52, and 0.57 ± 0.15 v. 3.58 ± 0.19; P < 0.05) and the rate of maturation (74.33% ± 4.32%, 57.83% ± 7.14%, 44.00% ± 5.93% v. 84.50% ± 3.62%; P < 0.05) in a dose-dependent manner during in vitro maturation for 44 h. Cell cycle analysis showed that NaF exposure blocked meiotic resumption, disturbed spindle dynamics, disrupted chromosome separation, and increased aneuploidy in porcine oocytes. Moreover, NaF exposure disturbed mitochondrial function, triggered DNA damage response, and induced early apoptosis in porcine oocytes. Exposure to NaF also induced oxidative stress, decreased GSH level, and increased cathepsin B activity in and impaired the further development potential of porcine oocytes, as indicated by a decrease in blastocyst formation rate, increase in apoptosis, and inhibition of cell proliferation. Together, these results indicate that NaF exposure impairs the maturation capacity of porcine oocytes by inhibiting cumulus cell expansion, disturbing cytoskeletal dynamics, and blocking nuclear and cytoplasmic maturation, thus decreasing the quality and affecting the subsequent embryonic development potential of porcine oocytes.

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DMBA acts on cumulus cells to desynchronize nuclear and cytoplasmic maturation of pig oocytes

Cited by 25 publications

References 44 publications

An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos

An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos

Ascorbic acid induces global epigenetic reprogramming to promote meiotic maturation and developmental competence of porcine oocytes

157 Sodium Fluoride Exposure Exerts Toxic Effects on Porcine Oocyte Maturation

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