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

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.


IntroductIon
In mammalian females, oocytes are arrested at the germinal vesicle (GV) stage of meiosis I and are stored in ovarian follicles for years or even decades. These GV stage-arrested oocytes have a low metabolic rate in order to maintain the stability of their inheritance materials (including genomic DNA and mitochondrial DNA [mtDNA] as well as other cellular organelles) and to accurately transmit them to the offspring [1]. During meiotic maturation, however, oocyte energy metabolism increases due to the requirements of multiple physiological events, such as GV breakdown (GVBD), spindle formation, chromosome alignment and separation, and polar-body extrusion (PBE) [2]. In addition, early embryonic development and implantation are also energyconsuming processes. Thus, numerous mitochondria develop and are stored in an oocyte's cytoplasm. They provide energy by producing ATP and by assisting with spindle assembly and orientation during meiotic maturation and early embryo cleavages [3,4].
Normal mitochondrial function is crucial for successful oocyte maturation and early embryonic development [5][6][7]. Mitochondrial dysfunction has been implicated in increased formation of abnormal spindles and chromosome aneuploidy in oocytes of mice fed a high-fat diet; these changes may account for the infertility observed in obese women [8,9]. Maternal diabetes results in defective oocyte meiosis by disrupting mitochondrial structures and metabolic functions [10]. Insulin www.impactjournals.com/oncotarget resistance was found to disrupt mitochondrial function by reducing mtDNA copy numbers and ATP levels in mouse MII oocytes. This mechanism may contribute to the low fertility rate in diabetic women [11]. Mitochondria aggregate in aged-oocyte cytoplasm and synthesize a reduced amount of ATP, and these defects can prevent oocyte maturation and ovulation and ultimately may result in female reproductive failure [5,12,13]. To date, however, there have been few studies on the regulation of mitochondrial dynamics during oocyte meiosis and embryonic development.
We recently identified a gene that encodes for a mitochondrial protein in Drosophila designated mitoguardin (Miga; Zhang and Liu et al., Molecular Cell, 2016 Jan, in press). It has two poorly studied homologs (family with sequence similarity 73, members A and B, Fam73a and Fam73b) in vertebrates, which we renamed Miga1 and Miga2. MIGA1 and MIGA2 are nucleusencoded proteins that are localized to the outer membrane of mitochondria. MIGA1/2 promote mitochondrial fusion by interacting with the mitochondrial outer-membrane protein Mito-PLD [14]. The latter is a signaling molecule involved in cardiolipin hydrolysis (in the synthesis of phosphatidic acid) and promotes mitochondrial fusion [14].
To identify the in vivo function of the Miga1/2, we generated Miga1 and Miga2 single-and double-knockout (KO) mouse strains and found that these KO females are subfertile. MIGA1/2 regulate mitochondrial dynamics and functions during oocyte meiosis and embryonic development. These results provide new insights into the mechanisms of mitochondrial fusion and may help to identify new therapeutic targets in female sterility of unknown etiology.

Inhibition of mitochondrial function blocked oocyte maturation
To assess mitochondrial function during oocyte meiosis, we incubated oocytes with the mitochondria-targeted compound carbonylcyanidem-chlorophenylhydrazone (CCCP), which is a proton gradient uncoupler, i.e., it disrupts mitochondrial ATP production. After CCCP treatment, oocytes meiotic maturation was arrested. More than 80% of the control oocytes resumed meiosis within 3 h of in vitro culture, and this process was characterized by GVBD, whereas oocytes treated with CCCP showed reduced GVBD rates, in a dose-dependent manner (in the range 1-10 μM CCCP; Figure 1A and 1B). In addition, CCCP-treated oocytes failed to release polar body 1 (PB1; Figure 1A and 1B) and degenerated in a dose-dependent manner ( Figure 1A and 1C).
Staining with fluorescent probes showed that mitochondria in the oocytes became clustered after CCCP treatment (10 μM, 3 h), and reactive oxygen species (ROS) levels increased (1 μM, 16 h. Figure 1D and 1E). Meiotic spindle formation was also compromised by CCCP treatment ( Figure 1F-1G). CCCP reduced ATP content of oocytes after incubation for 3 h ( Figure 1H). Collectively, these results indicated that oocyte meiosis requires active ATP production by mitochondria.
To confirm the low quality of the oocytes from the KO mice and to rule out the effects of other factors in vivo, fully grown GV stage oocytes were collected from antral follicles and cultured in vitro. Oocytes from the KO mice showed slightly decreased GVBD rates, after 3 h of culture ( Figure 2C and 2E). In line with the in vivo results, PB1 extrusion rates were significantly lower in KO oocytes than in WT oocytes after 16 h of culture ( Figure 2D and 2E). The oocytes from Miga1/2 KO mice had a phenotype (low PBE rate and high degeneration rate) similar to that of the oocytes treated with CCCP in vitro, suggesting that the Miga1/2 KO mice have low quality of oocytes because of mitochondrial defects.

Miga1/2-deleted oocytes
Because Miga1 and Miga2 are important for mitochondrial functions in somatic cells, we assessed mitochondrial functions in the oocytes of Miga1/2 -/mice. Mitotracker staining showed that mitochondria in WT oocytes were evenly distributed throughout the oocyte cytoplasm but partially gathered around spindles ( Figure  3A). In contrast, in the cytoplasm of Miga1 -/-, Miga2 -/-, and Miga1/2 -/oocytes, mitochondria were aggregated. These morphological changes resulted in increased ROS levels ( Figure 3A and 3B). ROS signals were colocalized with mitochondria, suggesting that ROS that were generated in the mitochondria accumulated and were caged in the mitochondria, thereby possibly adversely affected mitochondrial functions or ultrastructures.
High levels of ROS can cause damage to the mitochondria [15,16], and the decreased MMP usually correlates with alteration of the mitochondrial cristae structure [17]. Electron-microscopic images showed that in WT oocytes, mitochondria were generally evenly distributed with normal aligned cristae. In Miga1/2 -/oocytes, however, mitochondria were tethered closely to each other ( Figure 3E). More mitochondria lost their cristae and contained large vacuoles than did mitochondria in WT oocytes (43.3% in Miga1/2 -/oocytes versus 25% in WT oocytes, Figure 3F).  The mtDNA copy number is also an indicator of mitochondrial activity. We quantified mtDNA copy numbers in WT oocytes and embryos at the GV, MII, 1-cell, and 2-cell stages and found that mtDNA copy numbers increased nearly 2-fold from the GV to MII stage ( Figure 3G). The mtDNA copy numbers in MII-arrested Miga1/2 -/oocytes were approximately half of those in WT oocytes ( Figure 3H). In addition, ATP production in Miga1/2 -/oocytes was correspondingly reduced ( Figure  3I).

Precision of chromosome separation was disrupted in Miga1/2-deleted oocytes
Miga1/2 -/oocytes showed decreased PBE rates ( Figure 2C and 2F-2G) during meiotic maturation. Unexpectedly, most Miga1/2 -/oocytes could form spindles although they appeared thicker than those of WT oocytes ( Figure 4A and 4B). However, 63.6% of oocytes ovulated by Miga1/2 -/mice had abnormalities in the chromosome number or configuration, whereas only ~12.5% of chromosomes were abnormal in WT oocytes ( Figure  4C and 4D). Normal chromosomes in MII stage oocytes can be defined as 20 pairs of sister chromatids that were attached at centromeres; in contrast, in Miga1/2 -/oocytes, chromosome numbers ranged from 14 to 18 pairs. Some Miga1/2 -/oocytes were arrested at the MI stage with 20 pairs of homologous chromosomes (i.e., 80 chromatids; Figure 4C).

Miga1/2-deleted oocytes have a poor development potential after fertilization
The zygotes derived from Miga1 -/-, Miga2 -/-, and Miga1/2 -/oocytes all had a poor developmental potential as compared to embryos from WT females ( Figure  5A-5C). Although 72.8% of zygotes of WT females developed to blastocysts on day 4 after coitus, this rate was only 30.4% in Miga1/2 -/females ( Figure 5C). Most of the abnormal embryos degenerated, particularly during the development from the 1-cell to 4-cell stage ( Figure  5A). Mitochondria were clustered in the cytoplasm of the embryos from Miga1 -/-, Miga2 -/-, and Miga1/2 -/mice, especially in the embryos arrested at the morula stage, according to immunostaining for the mitochondrial protein HSP60 ( Figure 5D). These results indicated that the abnormal mitochondrial distribution in blastomeres that is caused by maternal deletion of MIGA1/2 may contribute to the failure of subsequent embryonic development.

AtP and vitamin c (Vc) partially reversed the defects of Miga1/2-deleted oocytes
Vc is an antioxidant that can reduce ROS levels in plant cells [18][19][20] and mouse embryonic fibroblasts [21]. Because deletion of MIGA1/2 in oocytes caused mitochondrial damage and disrupted mitochondrial dynamics, thus resulting in lower ATP levels and ROS accumulation in oocytes, we tested whether these defects could be reversed by Vc, at least partially. Vc treatment (25 μg/mL) increased PBE rates in both WT oocytes and Miga1/2 -/oocytes, but more significantly in the latter ( Figure 6A and 6B). In addition, Vc treatment remarkably reduced ROS levels and partially rescued the normal mitochondrial distribution in Miga1/2 -/oocytes ( Figure 6C and 6D). Meanwhile, ATP supplementation also partially reversed the PBE defects and reduced ROS levels in Miga1/2 -/oocytes ( Figure 6A-6C).

oocyte meiosis was arrested in conditional knockout mice
To determine whether the fertility in Miga1/2 -/mice was reduced by oocyte-related factors, we generated oocyte-specific Miga2 KO mice (Miga2 flox/flox Gdf9-Cre mice) and analyzed the oocyte meiosis in vitro. Oocytes from the Miga2 flox/flox Gdf9-Cre mice also contained aggregated mitochondria ( Figure 7A) and had defects in GVBD and PBE that were similar to those in the Miga2 -/oocytes ( Figure 7B and 7C). In addition, superovulation in Miga2 flox/flox Gdf9-Cre mice still produced a reduced number of ovulated oocytes ( Figure 7D), suggesting that it was the oocyte degeneration that contributed to the lower number of superovulated oocytes in Miga2 flox/flox Gdf9-Cre mice.

dIscussIon
Mitochondria undergo frequent morphological changes because of fission and fusion. Malfunctioning of the mitochondrial fusion and fission causes various human developmental disorders, including reduced fertility. Mitochondrial functions strongly correlate with mitochondrial structure and morphology [22,23]. When metabolism requires that mitochondria produce large amounts of ATP, mitochondria form wide long cristae, and mitochondria become elongated and connected in a persistent rapidly dynamic manner. In contrast, when mitochondrial functions are disrupted or slowed down (lowered metabolic rate), mitochondria form narrow short cristae and become fragmented or clustered depending on the cell type [24,25].
In a recent study (Zhang et al., manuscript submitted), we uncovered an evolutionarily conserved new family of outer-membrane mitochondrial proteins: MIGA, which promotes mitochondrial fusion in both www.impactjournals.com/oncotarget  In the present study, we further demonstrate that MIGA1 and MIGA2 are involved in oocyte meiosis, maturation, and developmental potency.
The mitochondria-targeted compound CCCP uncouples the MMP, disrupts ATP production, and results in mitochondrial fragmentation; these effects are similar to the phenotypes observed after deletion of Miga1/2 in mouse embryonic fibroblasts [16,26]. In the present study, CCCP arrested oocyte meiosis at the MI stage by inducing oocyte degeneration after oocyte GVBD. CCCP treatment strongly reduced ATP synthesis; in addition, elevated amounts of ROS were detected in clustered mitochondria, and this change contributed to abnormal spindle formation in the oocytes incubated with CCCP. The phenotype of Miga1/2 -/oocytes was similar to that of CCCP-treated oocytes in vitro, indicating that MIGA1/2 may regulate mitochondrial activities by controlling mitochondrial morphological remodeling.
The Miga1/2 -/oocytes showed defective mitochondrial dynamics and clustered mitochondria in the cytoplasm; these changes were probably linked to the high ROS levels inside these mitochondria. This high concentration of ROS may have reduced the mitochondrial membrane potential, reduced mtDNA copy numbers, and  Oncotarget 1165 www.impactjournals.com/oncotarget disrupted mitochondrial metabolism (ATP production). Furthermore, the lack of ATP may have contributed to the severe damage to the ultrastructures of mitochondrial cristae because most mitochondria lost their cristae and contained large vacuoles. PBE requires energy for separation of chromosomes and for division of the cytoplasm into 2 parts. The lack of ATP and an environment with a high ROS level may contribute to the failure of oocyte PBE. In addition, strong oxidative stress, such as that caused by H 2 O 2 , has been implicated in disruption of spindle formation, particularly during the MII stage of oocytes, by reducing the amount of mitochondria-derived ATP [27]. Once spindle formation or movement is disrupted, this change is very likely to cause abnormal chromosome separation and to increase the chances of aneuploidy. Additionally, ROS stress can reduce mtDNA copy numbers, thereby decreasing the ATP level and producing mutations in mtDNA [28,29].
Vc is an efficient antioxidant and can reduce high ROS concentrations [20,21]. We demonstrated that Vc treatment of oocytes reduced their ROS levels and partially reversed the defects in oocyte meiosis. Although Vc rescued mitochondrial morphology, it only partially reversed the defect in oocyte PBE in Miga1/2 -/mouse oocytes ( Figure 7E). These results suggest that the disordered mitochondrial dynamics that resulted in the defective PBE cannot be completely reversed simply by reducing ROS levels.
In summary, we demonstrated that the nucleusencoded mitochondrial proteins MIGA1 and MIGA2 are required for mitochondrial dynamics and functions in oocytes, and promote their developmental potential. Therefore, this study not only provides evidence of the physiological importance of mammalian MIGA proteins but also provides new insights into female infertility.

Mice
Miga1 KO mice were produced by TALEN from the FVB/N strain as described previously [30]. Miga2 KOfirst mice were purchased from the Jackson Laboratory.

oocyte culture
Twenty-one-day-old females were injected with 5 IU pregnant mare serum gonadotropin (Ningbo Sansheng Pharmaceutical Co., Ltd., China), and after 44 h, the mice were euthanized and the ovaries were chopped in a culture dish. Oocytes at the GV stage were cultured in drops of the M16 medium (M7292; Sigma-Aldrich) covered with mineral oil (M5310; Sigma-Aldrich) at 37°C in a humidified atmosphere containing 5% of CO 2 .

ros detection
ROS were detected by means of the ROS detection assay kit (Beyotime) according to the manufacturer's instructions. In short, oocytes were stained in 2',7'-dichlorofluorescin diacetate (DCFH-DA) in the M2 medium for 20 min at room temperature, washed, mounted on a glass slide, and examined under a confocal laser scanning microscope (Zeiss LSM 710, Carl Zeiss AG, Germany).

An immunofluorescence assay of the mouse oocytes
Oocytes were fixed in 4% paraformaldehyde (PFA) in PBS and incubated with 0.2% Triton X-100 in PBS for 30 min. After blocking the cells with 1% BSA in washing buffer (PBST: PBS with 0.1 % Triton X-100), we incubated oocytes with primary antibodies buffered in the blocking solution. After three washes, oocytes were incubated with secondary antibodies and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The oocytes were mounted on glass slides using SlowFade ® Gold Antifade Reagent (Life Technologies) and examined under a confocal microscope.

superovulation and fertilization
For superovulation, female mice (21-23 days) were injected intraperitoneally with 5 IU pregnant mare serum gonadotropin and 44 h later with 5 IU hCG (Ningbo Sansheng Pharmaceutical Co., Ltd., China). After 16 h, cumulus cell-oocyte complexes were excised from the ampullar region of oviducts and the numbers of oocytes were counted. The oocytes were examined and photographed by means of a Nikon SMZ1500 stereoscope.
To obtain early embryos, female mice were mated with 10-to 12-week-old WT males overnight. Successful mating was confirmed by the presence of vaginal plugs. Zygotes and 2-cell and 4-cell embryos were harvested