Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption
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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.
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.
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).
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.
KeywordsOocytes DNA damage DNA repair In vitro maturation Germinal vesicle
- 3.Dal Canto M, Brambillasca F, Mignini Renzini M, Coticchio G, Merola M, Lain M, et al. Cumulus cell-oocyte complexes retrieved from antral follicles in IVM cycles: relationship between COCs morphology, gonadotropin priming and clinical outcome. J Assist Reprod Genet. 2012;29(6):513–9.PubMedCentralCrossRefPubMedGoogle Scholar
- 7.Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. Nature Publishing Group; 2009 Oct 12;461(7267):1071–8Google Scholar
- 9.McDougall A, Elliott DJ, Hunter N. Pairing, connecting, exchanging, pausing and pulling chromosomes. 2005;120–5.Google Scholar
- 10.Govindaraj V, Basavaraju RK, Rao AJ. Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats. Reprod Biomed Online. Reproductive Healthcare Ltd; 2015;30(3):303–10.Google Scholar
- 27.Marangos P, Carroll J. Oocytes progress beyond prophase in the presence of DNA damage. Current Biology. Elsevier Ltd; 2012;22(11):989–94.Google Scholar