An aggregated mitochondrial distribution in preimplantation embryos disrupts nuclear morphology, function, and developmental potential

Abstract

A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function.

Keywords: Meiosis; Mitosis; Nuclear morphology; TRAK2; mitochondrial aggregation.

Fig. 1.

Exogenous Trak2 expression promotes bidirectional mitochondrial trafficking in GV oocytes. (A) Time-lapse images of two distinct types of mitochondrial redistribution after myc-Trak2 injection at t = 0. Time projection images are shown with spectrum pseudocolors (four different time points at 0, 1, 2, and 3 h). Type1: Mitochondria aggregation around the GV, Type2: Mitochondria aggregation at the oocyte cortex. (B) Representative images of mitochondrial redistribution induced by Trak2 overexpression. (C) The proportion of Trak2-expressing oocytes exhibiting Type1 and Type2. (D and E) Quantification of the number and size of mitochondrial clusters. Unpaired t test. (F) Representative images of mitochondrial redistribution in Trak2-expressing GV oocytes obtained from 3- and 6-wk-old mice. (G) Measurement of oocyte diameter obtained from 3- and 6-wk-old mice. Unpaired t test. (H) The proportion of Trak2-expressing oocytes from 3- and 6-wk-old mice showing Type1 and Type2. Chi-square. (I) Correlation between oocyte diameter and the area of mitochondria located in the cortex, excluding the perinuclear region. n = 171. Kolmogorov–Smirnov test. Oocytes were collected from 3-wk-old mice for (A–I) or 6-wk-old mice for (F–I). Scale bar is 15 μm (A), 10 μm (B) or 50 μm (F). n.s., not significant. ****P < 0.0001.

Fig. 2.

Trak2 overexpression does not alter mitochondrial MMP. (A) Representative examples of TMRM and mito-Dendra2 in control and Trak2-expressing oocytes. Psuedocolor ratiometric images of TMRM and mito-Dendra2 are shown. (B) Quantification of the fluorescence intensity ratio of TMRM to mito-Dendra2. The data were normalized by dividing the values of each group by the mean of control oocytes. Unpaired t test. Oocytes were collected from 3-wk-old mice. The scale bar is 15 μm (A). n.s.; not significant.

Fig. 3.

Mitochondrial aggregation by Trak2 delays meiotic resumption but not progression to MII. (A) A schematic diagram illustrating the analysis performed. (B) The percentage of oocytes undergoing NEBD. Chi-square. (C) The percentage of MI oocytes extruding the first polar body. Chi-square. (D) Representative images of mitochondrial distribution and MI spindles in control and Trak2-expressing oocytes. Dashed lines: Oocyte membrane. Yellow lines: Spindle-associated mitochondrial clusters. White arrowheads: Cytoplasmic mitochondrial cluster. (E) Ratiometric analysis comparing spindle-associated mitochondrial clusters to total mitochondrial clusters. Unpaired t test. (F) Measurement of MI spindle length. Unpaired t test. (G) Representative images of mitochondrial distribution and chromosomes in control and Trak2-expressing MII oocytes. Dashed line: Oocyte membrane. White arrow: Cortically localized mitochondria. (H) The percentage of oocytes showing cortically localized mitochondria. Chi-square. (I) Measurement of MII spindle length. Unpaired t test. (J) The number of chromosomes in control and Trak2-expressing MII oocytes. Unpaired t test. Oocytes were collected from 3-wk-old mice for the experiments. The scale bar is 10 μm (D and G), or 5 μm (G, chromosome spreads). n.s., not significant. **P < 0.01 and ****P < 0.0001.

Fig. 4.

In zygotes Trak2 induces a ball-like mitochondrial aggregate that self-centers in the cytoplasm. (A) Representative images (12 stacks of z-projection) of mitochondria in control and Trak2-expressing zygotes. (B) Schematic illustrating the method for measuring cytoplasmic mitochondrial distribution. Magenta: Mitochondria, Blue: PN, Yellow: ROI indicating where mito-Dendra2 were measured. (C) Measurement of normalized mito-Dendra2 intensity. Multiple unpaired t test. (D) Representative images of control and Trak2-injected zygotes showing TMRM and mito-Dendra2. The Right hand panel shows a pseudocolored ratiometric image obtained from TMRM and mito-Dendra2. (E and F) Quantification of the TMRM to mito-Dendra2 ratio at zygotes and two-cell embryos. The data were normalized by dividing the values of each group by the mean of the control. Unpaired t test. Zygotes were collected from 3-wk-old mice. The scale bar is 15 μm (A and D). n.s. not significant. *P < 0.05 ~ ****P < 0.0001.

Fig. 5.

Trak2-induced mitochondrial aggregation inhibits preimplantation development. (A) Trak2-injected zygotes were cultured to the two-cell stage, and the percentage of two-cells forming blastocysts is shown. Chi-square test. (B) Differential interference contrast images of embryos at 3.5 dpc. (C) Representative images of the first mitotic spindles in control and Trak2-expressing zygotes. (D) Measurement of the width of the spindle in the zygote after Trak2 expression. Unpaired t test. (E) Time-lapse images of chromosomes (H2B-mCherry) and mitochondrial (mito-Dendra2) distribution during the first mitotic division on the single plane. Note the chromosomes center within the mitochondrial mass during metaphase. Time is presented in minutes relative to chromosome condensation. (F) The first mitotic spindle formation in control or Trak2-injected zygotes. Dashed line; position of the mitochondrial aggregate. Top panel: a control zygote. Middle panel: a zygote in which two PN are separated by the mitochondrial aggregate. Bottom panel: a zygote in which two PN are located close to each other at the edge of the mitochondrial aggregate. Bottom Left panel: higher magnification of regions in white squares to better illustrate the merging of prespindles to form a single metaphase spindle. Time: minutes relative to the first image after NEBD. Maximum projection is shown of 18 z-scans at 2.5-um intervals. (G) Quantification of time from prespindle formation to chromosome aggregation. Unpaired t test. Zygotes were collected from 3-wk-old mice. The scale bar is 15 μm (D and F). n.s. not significant. ****P < 0.0001.

Fig. 6.

The nucleus and the mitochondrial aggregate induced by Trak2 compete for the central position in two-cell embryos. (A) Representative images of mitochondrial and nuclear localization in live (Top) and fixed (Bottom) two-cell embryos. (B) A schematic diagram illustrating the analysis performed to measure blastomere size in (C) and nuclear displacement in (D). (C) Measurement of blastomere size in control and Trak2-expressing two-cell embryos. Coefficient of variation, F-test to compare variances. (D) Measurement of the distance between the centroids of the nucleus and the blastomere. Unpaired t test. (E) Time-series images of nuclear positioning in Trak2 expressing two-cell embryos after treatment with Cytochalasin D (CCD) or/and Nocodazole (Noco). The nuclear position at the start of the 4 h imaging period is shown in the first and last images of each time series. (F) Measurement of normalized displacement of the nucleus from the initial localization (0 h). Simple linear regression. Data are represented as mean ± SD. Slope value in DMSO −0.025, CCD 0.029, Noco −0.07, and CCD+Noco 0.003. (G) Representative images of actin filaments in control and Trak2-expressing two-cell embryos after treatment with or without CCD. (H) Measurement of the cytoplasmic actin intensity. Unpaired t test. Zygotes were collected from 3-wk-old mice. The scale bar is 20 μm (A and G). ***P < 0.001 and ****P < 0.0001.

Fig. 7.

Trak2-induced mitochondrial mass distorts nuclear morphology. (A) Representative images of nuclear structure in two-cell stage embryos. Note the presence of MN in the Bottom panel. (B) Schematic diagrams illustrating the measurement of the distance between the nuclear centroid and nuclear outline (r) and the perfect circle based on the average outline (R) of control one- and two-cell stage embryos and illustrating the measurement of ellipticity. (C) The displaced distance of the nuclear outline from the perfect circle was measured and shown as the SD. Unpaired t test. (D and E) Measurement of nuclear ellipticity in one- and two-cell stage embryos. Unpaired t test. (F) The percentage of two-cell stage embryos containing MN. Chi-square test. Zygotes were collected from 3-wk-old mice. The scale bar is 10 μm (A). *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 8.

Mitochondrial aggregation suppresses DNA synthesis and global RNA transcription in two-cell embryos. (A) Representative images of EdU signals in two-cell embryos. Dashed line: Cell membrane. (B) Normalized intensity of EdU signals. Unpaired t test. (C) Representative image of γH2AX signals in two-cell embryos. White line: Nuclear outline. (D) Normalized intensity of γH2AX signals. Unpaired t test. (E) Representative images of 5-EU signals in control and Trak2-expressing two-cell embryos. Dashed line: Cell membrane. (F) Normalized intensity of 5-EU signals. Unpaired t test. (G) Representative images of 5mC and 5hmC in control and Trak2-expressing two-cell embryos. (H and I) Normalized intensity measurements of 5mC and 5hmC, respectively. Unpaired t test. (J) Comparison of 5hmC/5mC ratio in two-cell stage embryo control and Trak2-injected embryos. Unpaired t test. Zygotes were collected from 3-wk-old mice. The scale bar is 20 μm (A and E) or 15 μm (G). n.s.; not significant. **P< 0.01, ***P < 0.001, and ****P < 0.0001.