Coping with DNA Double-Strand Breaks via ATM Signaling Pathway in Bovine Oocytes

Abstract

As a common injury almost all cells face, DNA damage in oocytes-especially double-strand breaks (DSBs), which occur naturally during the first meiosis phase (meiosis I) due to synaptic complex separation-affects the fertilization ability of oocytes, instead of causing cancer (as in somatic cells). The mechanism of oocytes to effectively repair DSB damage has not yet been clearly studied, especially considering medically induced DSBs superimposed on naturally occurring DSBs in meiosis I. It was found that maturation rates decreased or increased, respectively corresponding with overexpression or interference of p21 in bovine oocytes. At the same time, the maturation rate of bovine oocytes decreased with a gradual increase in Zeocin dose, and the p21 expression in those immature oocytes changed significantly with the gradual increase in Zeocin dose (same as increased DSB intensity). Same as p21, the variation trend of ATM expression was consistent with the gradual increase in Zeocin dose. Furthermore, the oocytes demonstrated tolerance to DSBs during meiosis I, while the maturation rates decreased when the damage exceeded a certain threshold; according to which, it may be that ATM regulates the p53-p21 pathway to affect the completion of meiosis. In addition, nonhomologous recombination and cumulus cells are potentially involved in the process by which oocytes respond to DSB damage.

Keywords: ATM; DNA double-strand breaks; bovine oocyte; in vitro maturation; p21.

Figure 1

Expression of fluorescence proteins of Venus and p21-Venus after transfection in HeLa cells. (A,a): HeLa cells transfected with Venus; (B,b): HeLa cells transfected with p21-Venus. Scale bar = 10 μm.

Figure 2

Effects of p21 on oocytes: (A) cRNA microinjection of Venus or p21-Venus and co-injection with p21-Venus cRNA and p21-Morpholino in oocytes under Fluorescence microscope; scale bar = 100 μM; (B) the effects of p21 overexpression on the PBEI(%); and (C) the effects of p21 interference on the PBEI(%). * indicates p < 0.05.

Figure 3

Effects of DNA double-strand breaks (DSBs) induced by Zeocin on bovine oocyte in vitro maturation (IVM): (A) immunofluorescence of γH2AX in the oocytes after bovine cumulus–oocyte complexes (COCs) treated with 10 μM Zeocin for 2 h, scale bar = 50 μM; (B) polar body excretion (PBEI) (%) of oocytes after bovine COCs were treated with different concentrations of Zeocin (10, 50, 351.68 or 1758.38 μM) for 22 h. ** indicates p < 0.01.

Figure 4

Relative mRNA expression levels of DNA DSBs repair genes in oocytes by qRT-PCR. After bovine COCs were treated with different concentrations of Zeocin (0 μM, 10 μM, 50 μM, 351.68 μM or 1758.38 μM) for 22 h, the oocytes at PBEI (“maturation”, green charts) and oocytes at GV (“immature”, orange charts) were collected to detect DNA DSBs repair genes. * indicates p < 0.05, ** indicates p < 0.01.

Figure 5

Relative mRNA expression levels of genes in oocytes by qRT-PCR after bovine denuded oocytes (Dos) and COCs treated with different concentrations of Zeocin (10 μM and 50 μM) for 2 h. * indicates p < 0.05, ** indicates p < 0.01.

Figure 6

Effects of the ataxic telangiectasia mutation (ATM)-specific inhibitor KU-55933 on bovine COCs: (A) PBEI% of bovine COCs with different concentrations of KU-55933 treatment for 22 h; (B) after bovine COCs were treated with 10 μM KU-55933 for 22 h, the oocytes at PBEI (“maturation”, green charts) and oocytes at GV (“immature”, purple charts) were collected, in order to detect the genes for oocyte quality evaluation and cell cycle. * indicates p < 0.05, ** indicates p < 0.01.