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Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes - PubMed

  • ️Tue Jan 01 2019

Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes

Gaku Shimoi et al. J Reprod Dev. 2019.

Abstract

Mammalian oocyte quality degrades over time after ovulation in vitro, which can cause fatal defects such as chromosomal aneuploidy. As various oocyte manipulations employed in assisted reproductive technology are time consuming, post-ovulatory aging is a serious problem to overcome in reproductive medicine or ova research. In this study, we investigated the effects of postovulatory aging on the incidence of chromosome aneuploidy during meiosis II, with a focus on the expression of functional proteins from the spindle assembly checkpoint (SAC). Chromosome analysis was used to assess the rate of aneuploidy in in vitro aged oocytes, or in early embryos derived from aged oocytes. Immunofluorescent staining was used to detect the localization of MAD2, which is a SAC signal that monitors the correct segregation of sister chromatids. Immunoblotting was used to quantify cohesin subunits, which are adhesion factors connecting sister chromatids at the metaphase II (MII) centromere. It was shown that post-ovulatory oocyte aging inhibits MAD2 localization to the sister kinetochore. Furthermore, oocyte aging prevented cohesin subunits from being maintained or degraded at the appropriate time. These data suggest that the destabilization of SAC signaling causes sister chromatid segregation errors in MII oocytes, and consequently increases the incidence of aneuploidy in early embryos. Our findings have provided distinct evidence that the post-ovulatory aging of oocytes might also be a risk factor for aneuploidy, irrespective of maternal age.

Keywords: Aneuploidy; Cohesin; MAD2; Postovulatory aging; Spindle assembly checkpoint.

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Figures

Fig. 1.
Fig. 1.

Summary of the molecular mechanism of spindle assembly checkpoint. MAD2, which binds with MAD1 on the kinetochore, monitors the connection between a spindle fiber and kinetochore. The MAD1-MAD2 complex serves as a catalyst to promote binding between a free MAD2 and CDC20. The MAD2-CDC20 complex forms a M-phase checkpoint complex (MCC) with other functional proteins, and suppresses the activity of APC/C. A fully connected spindle fiber on the kinetochore interrupts the signal from the spindle checkpoint. CDC20, released from MCC, behaves as an APC/C activator by binding with APC/C. Activated APC/C promotes ubiquitination of securin, which regulates separase activity. The separase activated by securin degradation cleaves REC8, which is contained in the cohesin complex. Finally, sister chromatids lose cohesin, an adhesion factor, and are segregated by tension from the spindle fiber.

Fig. 2.
Fig. 2.

Comparison of the frequency of NCAs between aged and fresh groups. (A) Frequency of NCAs in mouse oocytes. (B) Frequency of NCAs in mouse 8-cell embryos. (C) Normal chromosome spread in mouse oocyte (n = 20). (D) Chromosome spread which shows aneuploidy in mouse oocyte (n = 19). (E) Chromosome spread which shows diploidy in mouse oocyte (n = 40). Scale bar = 10 µm. * P < 0.05.

Fig. 3.
Fig. 3.

Spindle shape and chromosome alignment in mouse MII oocytes. (A) Fresh oocytes, which were immediately collected at 15 hours after hCG administration. (B, C) Aged oocytes, which were cultured for 12 h in medium following oocyte collection. (D–F) Higher magnified images of spindle (left) and chromosome alignment (right). White arrowheads indicate chromosome misalignment. Immunofluorescent staining of α-tubulin (green). DNA (blue) was stained with DAPI. Scale bar = 10 μm.

Fig. 4.
Fig. 4.

Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and α-tubulin (green). DNA (blue) was stained with DAPI. D-PBS, instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.

Fig. 5.
Fig. 5.

Localization and expression level of MAD2 in mouse post-ovulatory aged oocytes. (A, B) Immunofluorescent staining of MAD2 (red) and α-tubulin (green) in 12 h aged oocytes. DNA (blue) was stained with DAPI. (A) No signal of MAD2 on the misaligned chromosome. (B) MAD2 localization on the chromosome, despite spindle fiber connections to kinetochores. The white arrowheads indicate MAD2 localization at the chromosome kinetochore. Scale bar = 10 μm. (C) Western blotting and expression levels of MAD2 in aged oocytes.

Fig. 6.
Fig. 6.

Comparison of expression levels of cohesin subunits, securin and CDC20 in mouse oocytes. Relative expression of SMC1β (A), SMC3 (B), REC8 (C), securin (D), and CDC20 (E). (F) Western blotting for each protein in oocytes before (left) and after (right) activation. β-actin was used as a loading control. Bars with different superscripts indicate significant differences (P < 0.05). * P < 0.05.

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