Effects of the Insemination of Hydrogen Peroxide-Treated Epididymal Mouse Spermatozoa on γH2AX Repair and Embryo Development

BACKGROUND: Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excessive reactive oxygen species (ROS) generation. METHODS AND FINDINGS: We used exogenous ROS (H(2)O(2)) to simulate cryopreservation and examined DNA damage repair in embryos fertilized with sperm with H(2)O(2)-induced DNA damage. Sperm samples were collected from epididymis of adult male KM mice and treated with capacitation medium (containing 0, 0.1, 0.5 and 1 mM H(2)O(2)) or cryopreservation. The model of DNA-damaged sperm was based on sperm motility, viability and the expression of γH2AX, the DNA damage-repair marker. We examined fertility rate, development, cell cleavage, and γH2AX level in embryos fertilized with DNA-damaged sperm. Cryopreservation and 1-mM H(2)O(2) treatment produced similar DNA damage. Most of the one- and two-cell embryos fertilized with DNA-damaged sperm showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed γH2AX in the one- and four-cell embryos. CONCLUSIONS: γH2AX may be involved in repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa

Oxidative Stress Delays Prometaphase/Metaphase of the First Cleavage in Mouse Zygotes via the MAD2L1-Mediated Spindle Assembly Checkpoint

In zygotes, DNA damage delays the first cleavage to enable repair. Our previous study found that 0.03 mM hydrogen peroxide (H2O2) was the minimum concentration required for induction of oxidative DNA damage in mouse zygotes and that this represented the most similar situation to the clinical phenomenon. In this study, we quantified the cleavage rates of cells in blastocysts at different developmental stages, followed by immunofluorescence to detect activation of γ-H2A histone family member X (a marker of DNA damage) in zygotes to confirm that oxidative DNA damage was induced in H2O2-treated zygotes. Monitoring H3S10P (phosphorylation of Ser10 on histone H3; a prometaphase/metaphase marker) levels at different hour postinsemination revealed that treatment of zygotes with 0.03 mM H2O2 resulted in a prometaphase/metaphase delay. Furthermore, immunofluorescence staining for mitotic arrest deficient 2-like 1 and the protein kinase TTK, components of the spindle assembly checkpoint (SAC), suggested that this delay possibly involved SAC activation. These studies of the relationships between oxidative stress and SAC can promote the success rate of in vitro fertilization

Fluorescence intensity of γH2AX in nuclei among fresh control, H₂O₂ and cryopreserved sperm (A) and quantification (B).

<p>Data are mean±SD, *<i>P</i><0.05 compared with 0.1 mM H₂O₂. DAPI = staining of nuclei.</p

Fertilization <i>in vitro</i> and development of mouse embryos with fresh spermatozoa and sperm treated with 1-mM H₂O₂.

<p>Fertilization <i>in vitro</i> and development of mouse embryos with fresh spermatozoa and sperm treated with 1-mM H₂O₂.</p

Immunohistochemistry of γH2AX expression in embryos.

<p>Changes in γ-H2AX expression in embryos fertilized by A: fresh sperm and B: sperm treated with H₂O₂. PI = propidium iodide staining.</p

Apoptosis of mouse embryos of different developmental stages in the untreated control and the treated groups.

<p>Data are presented as mean±SD, hpi: hours post insemination. The apoptotic rate for each embryo was expressed as the percentage of apoptotic cell number relative to the total number of the embryo. Embryos fertilized with hydrogen peroxide-stressed sperm were defined as the treated group, and those fertilized with fresh sperm were defined as the untreated control group.</p>*<p>p>0.05, the treated group vs. the untreated control group, Chi square test was use to compare the composition difference of apoptotic cell number of the two groups.</p

Supercapacitive performance of single phase CuO nanosheet arrays with ultra-long cycling stability

Copper oxide nanofilms can be fabricated on Cu foam by a simple electrochemical anodization process. However, it is difficult to obtain single-phase nanofilms that consist only of Cu2O or CuO. In this work, we present a modified anodization process that includes (NH4)6Mo7O24·4H2O in the electrolyte solution, and prepare single-phase CuO nanofilms grown directly on Cu foam. The surface morphologies of the CuO nanofilms are greatly dependent on the concentration of (NH4)6Mo7O24·4H2O included in the electrolyte solution during the anodization process, and accordingly present nanodots, nanoflakes, nanosheets, and/or nanobelts. The synthesis mechanism for CuO nanofilms is discussed in detail. The as-fabricated single-phase CuO nanofilms can be directly employed as electrodes that exhibit good supercapacitive performance, with an areal capacitance greater than 600 mF cm-2 at a current density of 1 mA cm−2 in a 2 M KOH aqueous solution. Moreover, the single-phase CuO nanofilm electrodes also demonstrate excellent long term cycling stability with about 94% retention of the initial areal capacitance after 10,000 charge/discharge cycles. The results demonstrate that the CuO nanofilms prepared on Cu foam by our modified anodization process are promising electrode materials for high-performance flexible supercapacitors