Mitoguardin-1 and -2 promote maturation and the developmental potential of mouse oocytes by maintaining mitochondrial dynamics and functions

Abstract

Mitochondrial dynamics change mitochondrial morphological features and numbers as a part of adaptive cellular metabolism, which is vital for most eukaryotic cells and organisms. A disease or even death of an animal can occur if these dynamics are disrupted. Using large-scale genetic screening in fruit flies, we previously found the gene mitoguardin (Miga), which encodes a mitochondrial outer-membrane protein and promotes mitochondrial fusion. Knockout mouse strains were generated for the mammalian Miga homologs Miga1 and Miga2. Miga1/2-/- females show greatly reduced quality of oocytes and early embryos and are subfertile. Mitochondria became clustered in the cytoplasm of oocytes from the germinal-vesicle stage to meiosis II; production of reactive oxygen species increased in mitochondria and caused damage to mitochondrial ultrastructures. Additionally, reduced ATP production, a decreased mitochondrial-DNA copy number, and lower mitochondrial membrane potential were detected in Miga1/2-/- oocytes during meiotic maturation. These changes resulted in low rates of polar-body extrusion during oocyte maturation, reduced developmental potential of the resulting early embryos, and consequently female subfertility. We provide direct evidence that MIGA1/2-regulated mitochondrial dynamics is crucial for mitochondrial functions, ensure oocyte maturation, and maintain the developmental potential.

Keywords: Pathology Section; ROS; female infertility; mitochondrion; mtDNA copy number; oocyte meiosis.

Figure 1. Inhibition of mitochondrial function by carbonylcyanide-m-chlorophenylhydrazone (CCCP) blocks oocyte maturation

A. Representative images of CCCP-treated oocytes. Scale bar = 100 μm. B. Effects of CCCP on germinal-vesicle breakdown (GVBD) and polar-body extrusion (PBE) rates of cultured oocytes. C. Effects of CCCP on degeneration rates of cultured oocytes. D. Fluorescent staining showing increased reactive oxygen species (ROS) levels (green) and clustered mitochondria (red) in CCCP-treated oocytes. Scale bar = 50 μm. E. Quantification of ROS signals in oocytes. F. Fluorescent staining showing disrupted spindles (green) and clustered mitochondria (red) in CCCP-treated oocytes. Scale bar = 50 μm. G. The proportion of normal spindles in control and CCCP-treated oocytes, after 16 h of culture. H. ATP contents in control and CCCP-treated oocytes.

Figure 2. Miga1/2^−/− mice show reduced fertility and defects in oocyte development

A. Polar body 1 (PB1) extrusion (PBE) rates of the oocytes ovulated in mice with the indicated genotypes. Total numbers (n) of observed oocytes are indicated. B. Degeneration rates of oocytes ovulated in mice with the indicated genotypes. Total numbers (n) of oocytes observed are indicated. C.-D. GVBD and PBE rates of cultured oocytes isolated from the mice of the indicated genotypes. Total numbers (n)of cultured oocytes are indicated. E. Representative images of ovulated (the upper row) and cultured oocytes (the middle and lower rows) of the indicated genotypes. Scale bar = 100 μm.

Figure 3. Mitochondria in oocytes of Miga1/2 knockout mice show an altered distribution and impaired functions

A. Mitochondria distributions and reactive oxygen species (ROS) levels in oocytes isolated from mice of the indicated genotypes, and fixed at 3 h after GVBD. Scale bar = 50 μm. B. Quantification of ROS levels in oocytes of the indicated genotypes. C. The mitochondrial membrane potential (MMP) in oocytes isolated from mice of the indicated genotypes, according to JC-1 staining. Scale bar = 50 μm. D. Quantitative results on the relative MMP levels in oocytes of the indicated genotypes. E. Electron microscopy results showing that mitochondria had lost cristae and formed vacuoles in Miga1/2^−/− oocytes. F. Quantitative results on the numbers of mitochondria with vacuoles in wild-type (WT) and Miga1/2^−/− oocytes. G. Relative mtDNA copy numbers in WT oocytes at indicated stages. H.-I. Relative mtDNA copy numbers (H) and ATP levels (I) in WT and Miga1/2^−/− oocytes at the MII stage.

Figure 4. Spindle assembly and chromosome separation in wild-type (WT) and Miga1/2^−/− oocytes

A. Confocal microscopy images showing spindles in WT and Miga1/2^−/− oocytes after 16 h of culture. The arrow indicates polar body 1 (PB1). Scale bar = 50 μm. B. and C. The percentage of oocytes with normal spindles (B) and normal chromosome numbers (C) after 16 h of culture. Total numbers (n) of the examined oocytes are indicated. D. Representative chromosome spreads from WT and Miga1/2^−/− oocytes after 16 h of culture. Chromosome configurations are shown by immunofluorescent staining for TOP2 (red) and CREST (green). DNA was labeled by 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm.

Figure 5. Early embryos derived from Miga1/2^−/− oocytes have a low developmental potential

A. Early embryos derived from oocytes of the indicated genotypes. Scale bar = 100 μm. B. Proportions of the indicated embryos from female mice of the indicated genotypes at day 0.5, 1.5, 2.0 after mating with wild-type (WT) males. C. Proportions of 6- to 16-cell embryos, morulae, blastocysts, and degenerated embryos from female mice of the indicated genotypes on day 4 after mating with WT males. D. The distribution of mitochondria in embryos from female mice of the indicated genotypes on day 4 after mating with WT males, as indicated by immunofluorescent staining for HSP60. Scale bar = 50 μm.

Figure 6. Vitamin C partially reverses mitochondrial defects in Miga1/2^−/− oocytes

A.-B. Images (A) and polar-body extrusion (PBE) rates (B) of oocytes after 12 h of culture, with or without addition of vitamin C or ATP to the media. Scale bar = 100 μm. C. Relative reactive oxygen species (ROS) levels of oocytes after 12 h of culture, with or without addition of vitamin C or ATP to the media. D. Confocal microscopy results showing ROS signals and mitochondrial distributions in oocytes after 12 h of culture, with or without addition of vitamin C or ATP to the media. Scale bar = 50 μm.

Figure 7. Phenotype analyses of mice with an oocyte-specific Miga2 knockout

A. Mitochondrial distributions in germinal vesicle (GV) stage oocytes of wild-type (WT) and Miga2^flox/floxGdf9-Cre mice (oocyte-specific Miga2 KO), as indicated by immunofluorescent staining for HSP60. Scale bar = 50 μm. B. Oocytes of WT and Miga2^flox/floxGdf9-Cre mice after 4 and 16 h of culture. Scale bar = 100 μm. C. In vitro germinal-vesicle breakdown (GVBD) and polar-body extrusion (PBE) rates of oocytes from mice of the indicated genotypes. D. Average numbers of oocytes being ovulated by mice of the indicated genotypes. E. Summary: Deletion of Miga1/2 or CCCP treatment results in mitochondrial clustering, reduces ATP levels and mitochondrial DNA (mtDNA) copy numbers, and increases reactive oxygen species (ROS) levels in oocytes; these changes cause meiotic and developmental defects. Addition of vitamin C or ATP to the oocyte culture medium partially reverses these problems.