Instituto de Gentica Veterinaria Prof. Fernando N. Dulout IGEVET, UNLPCONICET, Facultad de Ciencias Veterinarias FCV, Universidad Nacional de La Plata UNLP, Calle 60 y 118 sn, La Plata 1900, Buenos Aires, Argentina.
Cell Biol Int. 2011 Jun;35(6):593-7. doi: 10.1042/CBI20100507.
The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dose-dependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P = 0.014 for Zn1; P<0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P = 0.0308), control and Zn2 (P = 0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P = 0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations 'protect' the integrity of DNA molecule and diminish the percentage of apoptotic CC.
本研究旨在探讨锌（Zn）浓度对体外成熟过程中卵丘细胞（CCs）的影响。为此，通过彗星试验评估了向培养基中添加不同 Zn 浓度[0（对照）；0.7μg/ml（Zn1）；1.1μg/ml（Zn2）和 1.5μg/ml（Zn3）]对 CCs 中 DNA 完整性的影响。此外，通过 Annexin 染色分析了早期细胞凋亡。结果显示，Zn 处理组的 CCs 呈现出与剂量相关的 DNA 损伤显著减少。彗星试验分析的尾部矩（TM）在无 Zn 培养的细胞中明显更高（对照组，P<0.01），而 Zn 处理组的细胞则较低（对照组：5.24±16.05；Zn1：1.13±5.31；Zn2：0.10±0.36；Zn3：0.017±0.06）。所有处理均与对照组有统计学差异（Zn1：P=0.014；Zn2 和 Zn3：P<0.01）。对照组的凋亡细胞频率较高（对照组：0.142±0.07；Zn1：0.109±0.0328；Zn2：0.102±0.013；Zn3：0.0577±0.019）。对照组与 Zn1（P=0.0308）、对照组与 Zn2（P=0.0077）、对照组与 Zn3（P<0.0001）、Zn1 与 Zn3（P<0.001）和 Zn2 与 Zn3（P=0.0004）之间存在统计学差异。Zn1 与 Zn2 之间无差异。总之，低浓度 Zn 增加了体外培养的 CCs 中的 DNA 损伤和凋亡。然而，适当的 Zn 浓度可“保护”DNA 分子的完整性，并降低凋亡 CCs 的百分比。
