人类未成熟卵母细胞中的双链DNA断裂与修复反应及其与减数分裂恢复的相关性。
Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption.
作者信息
Coticchio Giovanni, Dal Canto Mariabeatrice, Guglielmo Maria Cristina, Albertini David F, Mignini Renzini Mario, Merola Maria, Lain Monia, Sottocornola Manuela, De Ponti Elena, Fadini Rubens
机构信息
Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi, 24, Monza, Italy.
Center for Human Reproduction, New York, NY, USA.
出版信息
J Assist Reprod Genet. 2015 Oct;32(10):1509-16. doi: 10.1007/s10815-015-0547-6. Epub 2015 Aug 4.
PURPOSE
Only 50-60 % of immature human oocytes attain the mature stage in vitro. Such a deficiency may be a reflection of inadequate conditions of in vitro maturation (IVM) or a manifestation of intrinsic oocyte defects. In the present study, we explored the possibility that the DNA of immature oocytes may be damaged and that such a condition, or inability to trigger a repair action, is associated to germinal vesicle (GV) arrest.
METHODS
Immature oocytes (GV-stage oocytes) were obtained from women undergoing stimulated (Stim-C) or IVM (IVM-C) cycles. GV oocytes obtained from stimulated cycles were fixed for successive analysis either after recovery (T0) or following 30 h (T30) of culture if still arrested at the GV stage. Oocytes retrieved in IVM cycles were used only if they were found arrested at the GV stage after 30 h (T30) of culture. All oocytes were fixed and stained to detect chromatin and actin. They were also assessed for positivity to γH2AX and Rad51, markers revealing the presence of double-strand DNA breaks and the activation of a DNA repair response, respectively. Labelled oocytes were analysed using a Leica TCS SP2 laser scanning confocal microscope.
RESULTS
In Stim-C oocytes, γH2AX positivity was 47.5 and 81.5 % in the T0 and T30 groups, respectively (P = 0.003), while γH2AX-positive oocytes were 58.3 % in the IVM-C T30 group (Stim-C T0 vs. IVM-C T30, P = 0.178; Stim-C T30 vs. IVM-C T30, P = 0.035). Positivity for nuclear staining to Rad51 occurred in 42.1 and 74.1 % of Stim-C in the T0 and T30 subgroups, respectively (T = 0.006), while 66.7 % of IVM-C T30 oocytes resulted positive for a DNA repair response (Stim-C T0 vs. IVM-C T30, P = 0.010; Stim-C T30 vs. IVM-C T30, P = 0.345).
CONCLUSIONS
The present data document the existence of double-strand DNA breaks (DSBs) in human immature oocytes. Also, they are consistent with the hypothesis that insults to DNA integrity may be an important factor affecting meiotic resumption.
目的
在体外培养时，只有50%-60%的未成熟人类卵母细胞能够达到成熟阶段。这种不足可能反映了体外成熟（IVM）条件不充分，或者是卵母细胞内在缺陷的一种表现。在本研究中，我们探讨了未成熟卵母细胞的DNA可能受损，以及这种情况或无法触发修复作用与生发泡（GV）期停滞相关的可能性。
方法
从未成熟卵母细胞（GV期卵母细胞）取自接受促排卵（Stim-C）或IVM（IVM-C）周期的女性。从促排卵周期获得的GV期卵母细胞，如果在恢复后（T0）或培养30小时后（T30）仍停滞在GV期，则固定用于后续分析。仅当在IVM周期中培养30小时后（T30）发现卵母细胞停滞在GV期时，才使用这些卵母细胞。所有卵母细胞均固定并染色以检测染色质和肌动蛋白。还评估了它们对γH2AX和Rad51的阳性反应，这两种标志物分别揭示双链DNA断裂的存在和DNA修复反应的激活。使用徕卡TCS SP2激光扫描共聚焦显微镜对标记的卵母细胞进行分析。
结果
在Stim-C组卵母细胞中，T₀组和T₃₀组γH2AX阳性率分别为47.5%和81.5%（P = 0.003），而IVM-C组T₃₀组γH2AX阳性卵母细胞为58.3%（Stim-C组T₀组与IVM-C组T₃₀组比较，P = 0.178；Stim-C组T₃₀组与IVM-C组T₃₀组比较，P = 0.035）。Rad51核染色阳性率在Stim-C组T₀亚组和T₃₀亚组中分别为42.1%和74.1%（T = 0.006），而IVM-C组T₃₀组66.7%的卵母细胞DNA修复反应呈阳性（Stim-C组T₀组与IVM-C组T₃₀组比较，P = 0.010；Stim-C组T₃₀组与IVM-C组T₃₀组比较，P = 0.345）。
结论
目前的数据证明了人类未成熟卵母细胞中存在双链DNA断裂（DSBs）。此外，这些数据与DNA完整性受损可能是影响减数分裂恢复的重要因素这一假设一致。
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