Stag3 regulates microtubule stability to maintain euploidy during mouse oocyte meiotic maturation

Abstract

Stag3, a meiosis-specific subunit of cohesin complex, has been demonstrated to function in both male and female reproductive systems in mammals. However, its roles during oocyte meiotic maturation have not been fully defined. In the present study, we report that Stag3 uniquely accumulates on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Depletion of Stag3 by gene-targeting morpholino disrupts normal spindle assembly and chromosome alignment in oocytes. We also find that depletion of Stag3 reduces the acetylated level of tubulin and microtubule resistance to microtubule depolymerizing drug, suggesting that Stag3 is required for microtubule stability. Consistent with these observations, kinetochore-microtubule attachment, an important mechanism controlling chromosome alignment, is severely impaired in Stag3-depleted oocytes, resultantly causing the significantly increased incidence of aneuploid eggs. Collectively, our data reveal that Stag3 is a novel regulator of microtubule dynamics to ensure euploidy during moue oocyte meiotic maturation.

Keywords: Stag3; aneuploid egg; chromosome alignment; microtubule stability; spindle assembly.

Figure 1. Localization of Stag3 during mouse oocyte meiotic maturation

A. Mouse oocytes at GVBD, prometaphase I, metaphase I, anaphase I and metaphase II stages were immunolabeled with anti-Stag3 antibody (red) and counterstained with Hoechst (blue). Images were acquired under the confocal microscope. Scale bar, 20 μm. B. Metaphase I oocytes were double-stained with anti-Stag3 antibody (red) and anti-α-tubulin-FITC antibody (green) and then counterstained with Hoechst (blue). Scale bar, 10 μm.

Figure 2. Effects of Stag3 depletion on the spindle formation and chromosome alignment in mouse oocytes

A. Protein levels of Stag3 in control and Stag3-KD (MO injected) oocytes. The blots were probed with anti-Stag3 antibody and anti-β-actin antibody, respectively. B. Representative images of spindle morphologies and chromosome alignment in control and Stag3-KD oocytes. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles and counterstained with Hoechst to visualize chromosomes. Scale bar, 20μm. C. The rate of aberrant spindles was recorded in control and Stag3-KD oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance. D. The rate of misaligned chromosomes was recorded in control and Stag3-KD oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance.

Figure 3. Effects of Stag3 depletion on the acetylated level of α-tubulin in mouse oocytes

A. Representative images of acetylated α-tubulin in control and Stag3-KD oocytes. Oocytes were immunostained with anti-acetylated α-tubulin antibody and counterstained with Hoechst to visualize chromosomes. Scale bar, 20μm. B. The immunofluorescence intensity was recorded in control and Stag3-KD oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance.

Figure 4. Effects of Stag3 depletion on the microtubule resistance to nocodazole

A. Representative images of microtubules before and after 5 min of treatment with nocodazole in control oocytes. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize microtubules and counterstained with Hoechst to visualize chromosomes. Scale bar, 10 μm. B. Representative images of microtubules before and after 5 min of treatment with nocodazole in Stag3-KD oocytes. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize microtubules and counterstained with Hoechst to visualize chromosomes. Scale bar, 10 μm.

Figure 5. Effects of Stag3 depletion on the kinetochore-microtubule attachment in mouse oocytes

A. Representative images of kinetochore-microtubule attachments in control and Stag3-KD oocytes. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles, with CREST to visualize kinetochores, and counterstained with Hoechst to visualize chromosomes. Scale bar, 10 μm. B. The rate of defective kinetochore-microtubule attachments was recorded in control and Stag3-KD oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance.

Figure 6. Effects of Stag3 depletion on the generation of aneuploidy in mouse eggs

A. Representative images of euploid and aneuploid MII eggs. Chromosome spread was performed to count the number of chromosomes. Chromosomes were counterstained with PI. Scale bar, 5μm. B. The rate of aneuploid eggs was recorded in control and Stag3-KD oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance.