Mitochondrial Calcium Disorder Affects Early Embryonic Development in Mice through Regulating the ERK/MAPK Pathway

The homeostasis of mitochondrial calcium ([Ca2+]mt) in oocytes plays a critical role in maintaining normal reproductive cellular progress such as meiosis. However, little is known about the association between [Ca2+]mt homeostasis and early embryonic development. Two in vitro mouse MII oocyte models were established by using a specific agonist or inhibitor targeting mitochondrial calcium uniporters (MCU) to upregulate or downregulate [Ca2+]mt concentrations. The imbalance of [Ca2+]mt in MII oocytes causes mitochondrial dysfunction and morphological abnormity, leading to an abnormal spindle/chromosome structure. Oocytes in drug-treated groups are less likely to develop into blastocyst during in vitro culture. Abnormal [Ca2+]mt concentrations in oocytes hindered epigenetic modification and regulated mitogen-activated protein kinase (MAPK) signaling that is associated with gene expression. We also found that MAPK/ERK signaling is regulating DNA methylation in MII oocytes to modulate epigenetic modification. These data provide a new insight into the protective role of [Ca2+]mt homeostasis in early embryonic development and also demonstrate a new mechanism of MAPK signaling regulated by [Ca2+]mt that influences epigenetic modification.


Introduction
Female infertility has been a serious problem among women worldwide. Oocyte activation failure is one of the contributing factors to infertility [1,2] since a properly developed oocyte is decisive for the subsequent fertilization, implantation, and embryonic development [3,4]. Mitochondrial dysfunction in oocytes caused by obesity, diabetes, or aging has been related to the reduced reproductive capacity [5,6]. Pre-vious studies have shown that mitochondria exhibit typical maternal hereditary characteristics [7]; meanwhile, their abundant production of adenosine triphosphate (ATP) is required for the reproductive process such as [8,9].
Mitochondrial calcium homeostasis is associated with oocyte activation disorders when mitochondrial energy production is impaired by oxidative stress or energy deficiency. Through establishing in vitro cell models (knocking down the gatekeepers of the [Ca 2+ ] mt uniporters Micu1 and Micu2    2+ ] mt would delay oocyte meiotic maturation. Damaged mitochondrial function and delayed meiotic resumption were also observed in these models [5,10]. These results altogether highlight the role of [Ca 2+ ] mt in oocyte meiosis; in particular, a proper level of [Ca 2+ ] mt in oocytes at the GV stage can be a biomarker to assess oocyte quality [11], despite that its contribution to oocyte maturation is still unclear. Therefore, the depth of our understanding for how [Ca 2+ ] mt determines the quality of metaphase II (MII) oocytes and regulates early embryonic development requires further expansion.
As protein kinases to specific amino acids (e.g., serine, threonine, and tyrosine), mitogen-activated protein kinase (MAPK) families play an important role in complex cellular programs such as cell proliferation [12]. MAPK families have also been related to spindle assembly regulation and microtubule organization [13][14][15]. Two studies showed that abnormal MAPK signaling blocks the GV breakdown (GVBD) of both porcine and cattle oocytes [16,17]. Our previous study showed that the calcium-sensing receptor as a gonadotropin-regulated factor promotes the maturation of porcine oocytes in a MAPK-dependent manner [18]. However, the underlying mechanism of how [Ca 2+ ] mt correlates with MAPK signaling to regulate the subsequent development of oocyte is yet poorly explored.
Epigenetic regulation is equally important in regulating oocyte maturation and early embryonic development [19]. Previous studies have shown that aberrant intracellular energy levels can induce changes in epigenetic modifications [20]. The crucial role of DNA methylation in early embryonic development has been elucidated [21] since an abnormal DNA methylated regulation may lead to abnormal development of oocyte and subsequent embryonic cleavage [22]. Although the regulatory role of DNA methylation in embryonic development has been elucidated [23], the relationship between [Ca 2+ ] mt and DNA methylation has not been fully determined.

Discussion
Obese, diabetic, and aging women having lower rate of oocyte activation, altered preimplantation embryo development, and even lower pregnancy rates have been highlighted [26][27][28][29], and these disorders have been related to the mitochondrial dysfunction in oocytes [5], because the ATP production and oxidative phosphorylation are essential for reproductive cellular processes including meiotic maturation and postimplantation development [30]. Our previous stud-  (Figure 1(c)-1(f)), indicating that spermine-or Ru360-treated MII oocytes are available in in vitro models to study the impact of [Ca 2+ ] mt disorder. We further found that the spindle/chromosome structure was damaged in drug-treated MII oocytes (Figures 2(a)-2(d)), indicating that oocytes are in poor quality, also supporting our previous studies that the depletion of MCU markedly disrupts spindle formation during oocyte meiosis [10]. The generation of ATP is one of the basic functions of mitochondria, and its cytoplasmic level reflects the quality of matured oocytes [31,32]. We then studied the effects of spermine and Ru360 on mitochondrial function (e.g., mitochondrial membrane potential and ATP levels in MII oocytes). Both mitochondrial membrane potential and ATP levels in MII oocytes were decreased after treatments with spermine and Ru360. In addition, changes in mitochondrial   13 Oxidative Medicine and Cellular Longevity morphology were observed in drug-treated groups including decreased mitochondrial densities and downregulated genes which are related to mitochondrial morphodynamics (Figures 3(e) and 3(f)). Consistently, defection in [Ca 2+ ] mt homeostasis damaged the mitochondrial function of MII oocytes, followed by impaired competence developing into the embryo (Figure 4).
The [Ca 2+ ] mt homeostasis in meiotic maturation of oocytes has been substantiated to be dependent on MAPK signaling pathways. The MAPK family is involved in regulating meiosis recovery in the follicle before ovulation as a signaling molecule; to be more specific, Mapk3/1 (also commonly known as ERK1/2) plays a role in gonadotropininduced signal transduction, and its activation in cumulus cells is critical for gonadotropin-induced GVBD and granulosa cell proliferation [33]. In this study, MII oocytes display an aberrant expression of p-MAPK after treatment with spermine or RU360, and their inability to develop into the early embryo can be neutralized by extra treatment by either U0126 or curcumin, respectively (Figures 6(l) and 6(m) and Table 1). Collectively, oocytes with [Ca 2+ ] mt disorder having a lower rate of embryonic formation (Figures 4(a)-4(f)) are possibly caused by the perturbed MAPK/ERK signaling pathway.
Epigenetic inheritance is a key factor in mediating oocyte maturation and early embryonic development [34,35]. Previous studies have shown that both ATP and ROS are concomitantly produced in the mitochondria via the tricarboxylic acid cycle, of which disturbance results in aberrant epigenetic modifications [36]. Moreover, the modification of DNA plays roles in oogenesis and early embryogenesis [37,38]. In this study, disruption of global genomic DNA methylation and altered expression levels of individual genes related to DNA methylation in MII oocytes were observed after treatments with spermine or Ru360 (Figures 5(a)-5(e)).
The involvement of the MAPK/ERK signaling pathway in epigenetic regulation in the mouse brain is observed [39]. Whether the level of [Ca 2+ ] mt in oocytes is related to epigenetic modifications regulated by the activation of the MAPK signaling pathway remains unknown. Here, we observed a defective expression of 5-mC in drug-treated groups, while these epigenetic abnormalities can be reversed by U0126 or curcumin (  Oxidative Medicine and Cellular Longevity disorder affects early embryonic development through the MAPK/ERK signaling pathway, of which overactivation or inhibition compromises the DNA methylation ( Figure 8). In brief, for MII oocytes, a proper physiological level of [Ca 2+ ] mt is critical for its maturation and competence of developing into the embryo. In summary, our study highlights the role of [Ca 2+ ] mt in maintaining both mitochondrial function and competence to develop into the embryo for oocytes, which is fulfilled by mediating the MAPK/ERK signaling pathway and epigenetic modifications. Although the DNA methylation in oocytes with [Ca 2+ ] mt disorder has been investigated in the current study, other epigenetic modifications such as histone acetylation await further investigation.

Conclusion
Our research using oocytes with [Ca 2+ ] mt disorder as in vitro models proved that [Ca 2+ ] mt plays a role in maintaining mitochondrial function and regulating epigenetic modification for oocytes. In addition, epigenetic inheritance can be damaged by ATP defection and the perturbed MAPK/ERK signaling pathway, leading to an impaired competence of MII oocytes to develop into the embryo.

Oocyte Collection and Culture.
Germinal vesicle (GV) stage oocytes were collected from the 3-week-old ICR mice. 5 IU of pregnant mare serum gonadotropin (PMSG, Ningbo Second Hormone Factory, Ningbo, China) was injected into the mice 46-48 hours before all experiments. GV stage oocytes were released from the fully grown follicles into prewarmed M2 medium supplemented with 2.5 μM milrinone, and cumulus cells were removed by repeated pipetting. After microinjection or any specific treatment, oocytes were thoroughly washed with DPBS and cultured in M16 medium under mineral oil at 37°C in a 5% CO 2 atmosphere incubator during the GV to MII stages.

Parthenoactivation of MII Oocytes.
The activation medium was calcium-(Ca 2+ -) free HTF complemented with 10 mM strontium chloride (SrCl 2 ) and 5 μg/mL cytochalasin B [40]. After being thoroughly washed in activation medium for three times, MII oocytes were activated in activation medium for 2.5 hours and then in regular HTF without SrCl 2 for 3.5 hours at 37°C with 5% CO 2 . Oocytes were then transferred from activation medium to KSOM plus (+) amino acids (KSOM/AA) medium (EmbryoMax® KSOM + AA with D-glucose and phenol red, EMD Millipore, Biller-ica, MA, USA). Development of embryos to the 2-cell, 4-cell, morula, and blastocyst stages was assessed 24, 48, 96, and 120 hours, respectively, after the start of initial culture in KSOM/AA medium.

Immunofluorescence.
Mouse MII oocytes were fixed in 4% (w/v) paraformaldehyde for 40 minutes at room temperature and then washed three times (10 minutes each) in washing buffer (PBS containing 0.01% Triton X-100 and 0.1% Tween-20). The oocytes were then permeated in 1% Triton X-100/PBS at room temperature for 1 hour and washed three times (10 minutes each) in washing buffer. The oocytes were then blocked with blocking buffer (1% BSA/PHEM with 100 mM glycine) for 1 hour at 37°C. The oocytes were incubated at 4°C overnight with a primary antibody (anti-α-tubulin 1 : 8,000) diluted in blocking buffer. After washing three times with washing buffer for 5 minutes each, the oocytes were incubated at 37°C for 1 hour with a corresponding second antibody (1 : 100 dilutions, CW Biotech) and washed four times in washing buffer for 10 minutes each. Finally, DNA was stained with 4′6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc., Burlingame, CA, USA). The oocytes were then expanded on glass slides and examined by confocal laser scanning microscopy (FLUOVIEW FV1000, Olympus, Tokyo, Japan) using the FLUOVIEW Viewer (Olympus, Tokyo, Japan). The excitation lasers were set at 488 nm, and emission channels of 520 nm were used for green fluorescence detection.
For 5-methyl cytosine (5-mC) staining, washed oocytes were denatured by 2 N hydrochloric acid (HCl) for 30 minutes at room temperature first and then blocked in blocking buffer for 1 hour. After washing for three times by washing buffer, oocytes were incubated with a mouse anti-5-mC-FITC antibody (1 : 300, Abcam) at 4°C overnight. DNA was then stained with 4 ′ 6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc., Burlingame, CA, USA). Finally, the samples were mounted on glass slides and examined with a confocal laser scanning microscope (FLUO-VIEW FV1000, Olympus, Tokyo, Japan) by using the FLUOVIEW Viewer (Olympus, Tokyo, Japan). The excitation lasers were set as 488 nm. The fluorescence intensity of a single oocyte was determined by using EZ-C1 Free-Viewer (Nikon, Tokyo, Japan). Oxidative Medicine and Cellular Longevity for 20 minutes. After that, oocytes were washed with DPBS for three times. Oocytes stained with different dyes were subsequently observed by confocal laser scanning microscopy (Nikon A1R, Tokyo, Japan) and quantified using a NIS-Elements AR (Nikon Instruments, Tokyo, Japan). 5.6. Investigation of Mitochondrial Density. Mitochondrial density was quantified by the mitochondrial reactive dye MitoTracker (Green) (Beyotime Institute of Biotechnology, China). Oocytes were placed into M2 medium complemented with 5 μM MitoTracker (Green) for 30 minutes and thoroughly washed with DPBS for three times. Confocal laser scanning microscopy (Nikon A1R, Tokyo, Japan) was used to observe the oocytes, and an NIS-Elements AR (Nikon Instruments, Tokyo, Japan) was used to quantify mitochondria.

Measurement of Intracellular Levels of ROS and GSH.
Intracellular levels of ROS and GSH were measured as described previously [41]. In brief, oocytes were incubated in M2 medium supplemented with either 1 mmol/L 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (H2DCFDA) or 10 μmol/L 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (Cell-Tracker Blue) for 30 minutes at 37°C and washed with DPBS for three times afterwards. The fluorescence was measured under an epifluorescence microscope with a filter at 460 nm excitation for ROS and 370 nm excitation for GSH (DP72, Olympus, Tokyo, Japan). Finally, the fluorescence intensity of ROS and GSH was determined by using EZ-C1 Free-Viewer (Nikon, Tokyo, Japan).

Quantification of Mitochondrial Membrane Potentials.
A mitochondrial membrane potential assay kit (JC-1 dye, Beyotime Institute of Biotechnology, China) was used to measure mitochondrial membrane potentials (Δφm). Oocytes were stained with a working solution containing 10 μM JC-1 at 37°C in a 5% CO 2 atmosphere for 20 minutes, after which they were washed with DPBS to remove the surface fluorescence, followed by observation under a fluorescence microscope (Olympus IX73). Red fluorescence indicates activated mitochondria (J-aggregates), while green fluorescence indicates less activated mitochondria (J-monomers), and the ratio of J-aggregates to J-monomers is calculated as the Δφm value.
5.9. ATP Content Assays. The ATP content in each oocyte was measured using the Enhanced ATP Assay Kit S0027 (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. Different ATP standards were prepared, ranging from 0 to 40 pmol ATP. Oocytes were then treated with 20 μM of lysis buffer within a 0.2 mL RNA-free centrifuge tube, and lysed cells were centrifuged for 5 minutes at 4°C and 12,000 × g. All steps were conducted on ice unless otherwise stated. ATP detecting solution was then added to 96-well plates and was left at room temperature for 3 to 5 minutes. Standard solutions and ATP detection diluents were then added into each well. Samples were also added to each well, and the luminescence signals were immediately calculated with a luminometer (Infinite F200; Tecan). The ATP content of the samples was then calculated based on the standard curves. Total ATP levels were divided by the number of oocytes in each sample to calculate the mean ATP content per oocyte (pmol/oocyte). 5.10. Quantitative PCR. Total RNA was extracted using a RNeasy microRNA isolation kit (Qiagen, Valencia, CA, U.S.) following the manufacturer's instructions. Samples were treated with DNase I, and then, Transcript-Uni Cell was used for cDNA synthesis. A quantitative PCR supermix was used for the assays (Transgene Biotech, Beijing, China). RNA concentrations were measured using a Nanodrop 2000 Spectrophotometer (Biolab, Scoresby, Victoria, Australia) at a wavelength of 260 nm. Samples for subsequent analyses were only used if their 260 : 280 nm absorbance ratios were >1.8. Quantitative-and reverse transcription-PCR assays were performed with an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.), respectively. Three replicates were conducted for all assays. The relative expression of genes was calculated by the comparative threshold cycle method as 2 −ΔΔCt . The primers used for the amplification assays are shown in Table 2.
5.11. Statistical Analysis. Each experiment was repeated at least three times. A representative image of each experiment is shown. All data were analyzed using Student's t-test or one-way analysis of variance (ANOVA) examined by Duncan's multiple range test in SPSS software (IBM, Chicago, IL, USA). Data are expressed as the mean ± SEM.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Additional Points
Summary Statement. Mitochondrial Ca 2+ ([Ca 2+ ] mt ) plays a key role in early embryonic development. In this study, we firstly demonstrated that the ERK/MAPK signaling pathway is regulated by [Ca 2+ ] mt in MII oocytes, of which disorder influences the epigenetic modification and preimplantation development of the mouse embryo.

Conflicts of Interest
The authors declare no conflict of interest.

Authors' Contributions
Luyao Zhang participated in the research design, animal research, data analyses, and drafting of this article. Kexiong Liu participated in animal experiments. Qingrui Zhuan, Zhiqiang Liu, and Lin Meng participated in animal research and reviewed this article. Xiangwei Fu and Gongxue Jia participated in drafting and reviewing this article. Yunpeng Hou provided substantial advice in study design, assistance in division of labor, drafting, and revision. 16 Oxidative Medicine and Cellular Longevity