DNA double-stand breaks in normal, Zeiocin-treated, carbenoxolone (CBX)- treated and Zeiocin/CBX-double treated COCs.

<p>(A) Normal and zeiocin-treated COCs. (B) CBX-treated normal and DNA-damaged oocytes. γH2A.X, serine 139 phosphorylated histone H2A.X, a marker of DNA double-strand breaks.</p

Immunostaining of cAMP in DSB COCs.

<p>(A) cAMP enriched in the zona pellucida of DSB COCs. (B) Cumulus cells of COCs were removed by vibrating, and the zona pellucida was removed by acidic Tyrode solution. Compared with normal group, the nucleus of DSB oocytes enriched with cAMP. Blue, Hoechst; Green, γH2A.X; yellow, cAMP. DSB, DNA double-strand breaks.</p

DNA double-strand breaks (DSBs) up-regulate <i>Adcy1</i> in cumulus cells and down-regulate <i>Pde3a</i> in cumulus-enclosed oocytes.

<p>(A) <i>Adcy1</i> is up-regulated in the cumulus cells from cumulus oocyte complexes (COCs)with DNA DSBs.(B) The expression of <i>Gpr3</i> and <i>Pde3a</i> in oocytes from normal and DNA DSB COCs. (C)The expression of <i>Gpr3</i> and <i>Pde3a</i> in normal and DNA DSB denuded oocytes. **, highly significant difference, p < 0.01; *, significant difference, 0.01 < p < 0.05.</p

Germinal vesicle breakdown (GVBD) rates of denuded oocytes and cumulus enclosed oocytes (CEOs).

<p>(A) GVBD rates of denuded oocytes after 4 hours <i>in vitro</i> maturation (IVM). (B) GVBD rates of denuded oocytes after 15 hours IVM. (C) GVBD rates of CEOs after 4 hours IVM. COC, cumulus enclosed oocyte. Ctl, control or normal group; Zei, Zeiocin-treated group; CBX, carbenoxolone-treated group; Zei+CBX, Zeiocin/CBX-double treated group.</p

Effect of knock—down of <i>Adcy1</i> in cumulus cells on nuclear actin filament formation.

<p>(A) Efficiency of RNAi by <i>Adcy1</i> siRNA. (B) <i>Adcy1</i> siRNA transfection induced the decreasing of Adcy1 protein in cultured cumulus cells. (C) Upon knock-down of <i>Adcy1</i> mRNAs, there was no obvious change in the nuclear actin filament formation.</p

Blocking of gap junctions could induce nuclear actin filament formation in CEOs.

<p>When the CEOs were treated with the gap-junction inhibitor carbenoxolone (CBX), nuclear actin filaments formation was observed in oocytes. Corresponding to this, BLM-induced nuclear actin filaments were not inhibited by CBX.</p

Formation of nuclear actin filaments in DNA-damaged CEOs.

<p>Treatment of CEOs with BLM for 1 h led to actin filament formation in oocyte nuclei (marked by white asterisks in the zoom-in images from last panel).</p

Nuclear actin filaments are not formed in the DNA-damaged DOs.

<p>The DNA DSBs (marked by γH2A.X; green) were induced by 10 μM or 40 μM BLM. The actin filaments were labeled with phalloidin.</p

Inhibition of nuclear actin filament formation by Chek1/2 inhibitor, AZD7762 (AZD).

<p>No nuclear actin filament formation was observed upon treatment of CEOs with AZD alone. Treatment of CEOs with both BLM and AZD didn’t induce nuclear actin filament formation in oocyte nucleus.</p

Nuclear actin filament formation upon treatment of CEOs with BLM for different time points.

<p>The nuclear actin filaments could be detected in the CEOs that were treated with BLM for 1 h or 2 h, but not in the ones treated with BLM for 0.5 h.</p