Oxidative damage to mitochondrial DNA induced by arsenic trioxide in mouse oocytes in vitro and in vivo / 中国药理学与毒理学杂志

OBJECTIVE To investigate the arsenic trioxide(As2O3)-induced oxidative damage to mitochondrial DNA (mtDNA) in mouse oocytes and possible mechanisms. METHODS ① For in vitro assay,the mouse oocytes were denuded from ovaries of normal mice and incubated in medium for 20 h in different treatment groups:control,As2O3 1 and 2 μmol · L- 1,N-acetylcysteine (NAC) 5 mmol · L-1, 2,2,6,6-tetramethyl-1-piperidinyloxy(Tempo)1 mmol · L-1, As2O3(1 and 2 μmol · L-1)+NAC 5 mmol · L-1,As2O3 (1 and 2 μmol · L-1)+Tempo 1 mmol · L-1. ② For in vivo assay,mice were subjected to ip injection with physiological saline (normal control),As2O3 1 and 2 mg · kg-1,or As2O3 (1 and 2 mg · kg-1)+NAC 200 mg · kg-1, respectively. After 60 d,all the mice were sacrificed and their ovaries were quickly excised. Intracellular reactive oxygen species(ROS) levels were determined by 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The oxidative damage to mtDNA was induced using enzyme-linked immunosorbent assay(ELISA)for 8-hydroxy-2′-deoxyguanosine(8-OHdG). The expression of DNA polymerase γ(Polγ)and mitochondrial transcription factor A(mtTFA)was detected by Western blotting and the vitality of lysosomes was monitored by β-galactosidase(β-Gal)Assay Kit. RESULTS ①In vitro experiments,As2O3 elevated 8-OHdG levels of mtDNA in mouse oocytes accom? panied by increased levels of ROS (P<0.05),but co-treatment with NAC or Tempo significantly reduced ROS and 8- OHdG levels (P<0.05). Meanwhile, the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05),but were markedly elevated by the addition of NAC or Tempo (P<0.05). ②In vivo assay,As2O3 elevated ROS as well as 8-OHdG levels of mtDNA in mouse oocytes,while the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05). Co-treatment with NAC significantly reduced ROS and 8-OHdG levels,but markedly elevated Pol γ and mtTFA levels(P<0.05). Besides,a notable increase in β-Gal activity was shown in As2O3-treated mouse oocytes in vitro (P<0.05),while antioxidants efficiently reduced the activity (P<0.05). However,no significant changes were observed in the in vivo study. CONCLUSION The oxidative damage to mtDNA induced by As2O3 in mouse oocytes may be mediated by ROS and associated with down-regulation of protein levels of Pol γ and mtTFA as well as increment of lysosomal activity.

Damage of Intracytoplasmic Meiotic Spindles in Cryopreserved-Thawed Mouse Oocytes / 대한산부인과학회잡지

OBJECTIVE: To investigate the extent of meiotic spindle damages in cryopreserved-thawed mouse mature oocytes. METHODS: After slow freezing and ultra-rapid thawing using 1.5M dimethylsulfoxide(DMSO), mouse mature oocytes were stained by anti-alpha tubulin monoclonal antibody. The meiotic spindle and chromosomes configuration were assessed using confocal microscope. The influence of time to post-hCG oocytes retrieval (i.e. 12 hrs vs 17 hrs) was also evaluated. RESULTS: The normal meiotic spindles were observed in 89.8% of post-hCG 12 hrs group, and 80.1% of post-hCG 17 hrs group, and these were significantly lower than that of each unfreezed control. Post-hCG 12 hrs group showed a significantly higher incidence of normal meiotic spindles, compared with post-hCG 17 hrs group. CONCLUSION: The extent of meiotic spindle damages was significantly increased after cryopreservation in mouse mature oocytes. We proposed that 12 hrs interval of post-hCG oocytes retrieval may be more beneficial.

Establishment of Mouse Oocytes Cryopreservation Program / 대한산부인과학회잡지

OBJECTIVE: To establish the optimal cryopreservation method in mouse oocytes. METHODS: Firstly, mouse immature oocytes were exposed to various cryoprotectants, and then cryoprotectant with the best outcome was selected. Secondly, mouse immature oocytes were cryopreserved by either slow freezing and ultra-rapid thawing or vitrification. Finally, in mouse mature oocytes, the five different protocols were compared in their fertilization and hatching rates. RESULTS: 1) 1.5M 1,2-propanediol (PROH) and 1.5M PROH+0.1M sucrose had a higher rate of survival (73.1%, 81.9%) and in vitro maturation (28.2%, 30.1%). 2) Vitrification using 5.5M ethylene glycol (EG) showed significantly higher rate of survival and in vitro maturation, when compared with slow freezing and ultra-rapid thawing using 1.5M PROH+0.1M sucrose (65.9% vs 50.0%, 40.0% vs 28.2%, respectively). 3) In mouse mature oocytes, vitrification using 5.5M EG showed significantly higher survival rate, however, slow freezing and ultra-rapid thawing using 1.5M DMSO was superior to vitrification in view of fertilization rate. CONCLUSIONS: Vitrification showed better outcomes in mouse immature oocytes, but slow freezing and ultra-rapid thawing using 1.5M DMSO may be beneficial in mature oocytes.