MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes

Abstract

Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.

Keywords: APC/C-Cdh1; DNA damage; G2/M transition; MDC1; Oocytes; Spindle assembly.

Fig. 1

MDC1 is required for ATM-mediated DNA damage response in mouse oocytes. A GV oocytes were injected with either MDC1 siRNA (siMDC1) or control siRNA (siCTL) and cultured for 24 h in the presence of IBMX for knockdown. Depletion of MDC1 was confirmed by immunostaining with MDC1 antibodies. Scale bar, 10 μm. B Quantification of MDC1 levels after knockdown. Data are expressed as the mean ± SEM of three independent experiments. C–H After treating with etoposide (ETP) or DMSO for 30 min, oocytes were fixed and subjected to TUNEL assay or immunostaining with γ-H2AX and p-ATM antibodies. The fluorescence intensity of TUNEL, γ-H2AX and p-ATM were measured and shown with representative images. Scale bar, 10 μm. Data are expressed as the mean ± SEM of three independent experiments. ***P < 0.0001; ns, not significant

Fig. 2

MDC1 regulates G2/M transition by stabilizing cyclin B1 in mouse oocytes. A, B Control (siCTL) and siMDC1-injected oocytes were treated with etoposide (ETP). The incidence of GVBD for 4 h and the developmental stage of oocytes after 24-h culture were quantified. C Immunoblotting analysis of cyclin B1 and pY15-Cdk1 at 0 and 2 h after IBMX release in siCTL- or siMDC1-injected oocytes. D, E Levels of pY15-Cdk1 and cyclin B1 were normalized to β-actin, quantified, and expressed in arbitrary unit (a.u.). Data are expressed as the mean ± SEM from three independent experiments. *** P < 0.0001

Fig. 3

MDC1 regulates APC/C-Cdh1-mediated cyclin B1 degradation during G2/M transition. A The incidence of GVBD was quantified for 4 h after IBMX release in oocytes injected with siCTL, siMDC1 or siMDC1 + cyclin B1 mRNA. B–F Oocytes were injected with siCTL, siMDC1 and siMDC1 + Cdh1 MO. After 24-h culture, Cdh1 levels were determined using immunoblotting and immunostaining analyses. B, C Immunoblotting analysis of Cdh1. Levels of Cdh1 were normalized to β-actin, quantified, and expressed in arbitrary unit (a.u.). Data are expressed as the mean ± SEM from three independent experiments. D, E Immunostaining of Cdh1. The fluorescence intensity of Cdh1 was measured and shown with representative images. Scale bar, 10 μm. *** P < 0.0001. F The incidence of GVBD was quantified for 4 h after IBMX release

Fig. 4

MDC1 interacts with APC/C-Cdh1 in a DNA damage dependent manner. Oocytes were exposed to etoposide (ETP) and cultured in fresh medium for recovery for 1 h and 3 h. Oocytes injected with siMDC1 were used as a negative control. A In situ PLA of oocytes. B The number of PLA puncta per oocyte was quantified. C–E Immunostaining analysis of Cdh1 and MDC1. Scale bar, 10 μm. D The number of MDC1 foci was quantified. E The fluorescence intensity of Cdh1 and MDC1 were quantified. F Immunostaining analysis of cyclin B1. Scale bar, 10 μm. G The fluorescence intensity of cyclin B1 was quantified. * P < 0.05; *** P < 0.0001. H The incidence of GVBD was quantified for 4 h after IBMX release

Fig. 5

MDC1 is essential for MTOC organization during meiotic maturation. Oocytes were injected with cyclin B1 mRNA alone (CTL) or siMDC1 + cycin B1 mRNA (Cyclin B1 rescue). After 24-h culture, oocytes were released from IBMX. A Polar body extrusion (PBE) rate was scored after 16-h culture post-IBMX release. B–G After 8 h post-IBMX release, oocytes were fixed and immunostained with α-tubulin, ACA, PCNT, p-Aurora A or CEP192 antibodies. For ACA staining, oocytes were briefly placed on ice before fixation. Scale bar, 10 μm. Chromosome misalignment; spindle abnormality; kMT attachment; and intensity of PCNT, p-Aurora A, and CEP192 were quantified. (G) The number of MTOCs around chromosomes was quantified using CEP192 foci. * P < 0.05; ** P < 0.001; *** P < 0.0001

Fig. 6

Model of MDC1-mediated regulation of G2/M transition in response to DNA damage. A Working model for the functions of MDC1 during G2/M transition. In the normal state, MDC1 is associated with APC/C-Cdh1 and regulates cyclin B1 to maintain threshold levels. However, in the presence of DNA damage, MDC1 is dissociated from APC/C-Cdh1 and recruited to DNA damage sites. This activity increases APC/C-Cdh1-medidated cyclin B1 degradation. B Comparison of the G2/M DNA damage checkpoint between somatic cells and oocytes