Mitochondrial Calcium uniporters are essential for meiotic progression in mouse oocytes by controlling Ca2+ entry

Abstract Objectives The alteration of bioenergetics by oocytes in response to the demands of various biological processes plays a critical role in maintaining normal cellular physiology. However, little is known about the association between energy sensing and energy production with energy‐dependent cellular processes like meiosis. Materials and methods We demonstrated that cell cycle‐dependent mitochondrial Ca2+ connects energy sensing to mitochondrial activity in meiosis progression within mouse oocytes. Further, we established a model in mouse oocytes using siRNA knockdowns that target mitochondrial calcium uniporters (MCUs) in order to inhibit mitochondrial Ca2+ concentrations. Results Decreased numbers of oocytes successfully progressed to the germinal vesicle stage and extruded the first polar body during in vitro culture after inhibition, while spindle checkpoint‐dependent meiosis was also delayed. Mitochondrial Ca2+ levels changed, and this was followed by altered mitochondrial masses and ATP levels within oocytes during the entirety of meiosis progression. Abnormal mitochondrial Ca2+ concentrations in oocytes then hindered meiotic progress and activated AMP‐activated protein kinase (AMPK) signalling that is associated with gene expression. Conclusions These data provide new insight into the protective role that MCU‐dependent mitochondrial Ca2+ signalling plays in meiotic progress, in addition to demonstrating a new mechanism of mitochondrial energy regulation by AMPK signalling that influences meiotic maturation.

biological processes including differentiation, apoptosis and cell division, 4 while calcium signalling plays vital roles in cell cycle regulation. 5 Numerous studies have shown that there is a rapid rise in Ca 2+ levels in the cytoplasm during mitosis and oocyte maturation. 6 Oocytes spend a considerable amount of time having entered meiosis and are arrested at the first meiotic prophase. 7 In preparation for fertilization, mammalian oocytes undergo a process that causes dramatic increases in cytosol Ca 2+ levels. 8 Thus, these observations indicate that Ca 2+ transiently promotes meiotic progression.
Increased cytosolic Ca 2+ levels lead to rapid Ca 2+ uptake by mitochondria. Numerous studies have shown that mitochondrial calcium uniporters (MCUs) are unique channels that control calcium influxes. 9,10 Further, mitochondrial Ca 2+ levels promote the activities of the mitochondrial electron transport chain and the tricarboxylic acid (TCA) cycle, leading to ATP production. 11,12 Indeed, mitochondria are the primary sources of energy for ATP synthesis, which is required for numerous biological processes. 13 Importantly, abnormal accumulation of mitochondrial Ca 2+ can lead to mitochondrial dysfunction, decreased ATP production and other problems. 14 Consequently, ATP synthesis is an indicator of mitochondrial function, further highlighting the indispensable relationship between energy sources and mitochondrial function. 15 Meiotic progression demands high energy inputs to drive DNA synthesis and chromosome segregation, which are critical for oocyte maturation. 15,16 Therefore, a close association exists between mitochondrial Ca 2+ signalling and meiosis, although the underlying mechanisms are still unknown.
AMP-activated protein kinase (AMPK) signalling is a crucial cellular energy sensory mechanism. AMPK is a unique protein kinase that is regulated by the ATP/energy status and helps re-balance ATP production. 14 The kinase activity of AMPK complexes is instantaneously increased by acutely altered AMP levels, suggesting AMPK activation is due to ATP consumption and AMP accumulation. 17 ATP is mainly synthesized in oocytes via the mitochondria, which are the energy factories of cells. In this study, we investigated the key role of MCU-mediated mitochondrial Ca 2+ homeostasis in maintaining energy homeostasis during meiosis. In response to the low energy status during meiosis, the energy sensor AMPK phosphorylates and prevents meiotic maturation. Our results demonstrate a critical role for MCUdependent mitochondrial Ca 2+ signalling that connects energy sensing to proper meiotic progression and AMPK phosphorylation.

| Animal experiments
Mice were kept under 12-h/12-h light-dark cycles in a dedicated pathogen-free environment at the Central Animal Laboratory of the Institute of Zoology. All procedures were performed with the approval of the Institutional Animal Care and Use Committee of China Agricultural University (AW01040202-1).

| 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) was injected into the mice 46-48 h before all experiments. GV-stage oocytes were released from the fully grown follicles into pre-warmed M2 medium supplemented with 2.5 μM milrinone, and cumulus cells were removed by repeated pipetting. After microinjection or another specific treatment, oocytes were thoroughly washed with DPBS and cultured in MI6 medium under mineral oil at 37°C in a 5% CO 2 atmosphere incubator during the GV to MII stages.

| Measuring mitochondrial Ca 2+ ([Calcium] m )
[Calcium] m levels were measured using Rhod-2AM (Invitrogen/Molecular Probes, Carlsbad) according to the manufacturer's instructions. Zona pellucida was removed by pronase E. The oocytes were then stained with 5 μM Rhod-2AM for 30 min in maturation medium and thoroughly washed with DPBS, followed by incubation in maturation medium-free Rhod-2AM at 37°C under a 5% CO 2 atmosphere for 30 min. Cells were subsequently observed with confocal laser scanning microscopy (Nikon A1R) and quantified using a NIS-Elements AR (Nikon Instruments).

| Measuring cytosolic Ca 2+ ([Calcium] i )
Cytosolic Ca 2+ levels were assessed using Flou-3 AM (Invitrogen/ Molecular Probes, Carlsbad). First, zona pellucid was enzymatically removed by 0.5%pronase 37°C for 5 min. The oocytes were then processed in maturation medium with 5 μM Flou-3 AM for 40 min and washed three times by DPBS. Subsequently, they were analysed using a confocal laser scanning microscope (Nikon A1R) and quantitatively processed using NIS-Elements AR (Nikon Instruments).

| siRNA microinjection
Small interference RNAs (siRNA) for MCU (CCAAAGAG ACCU AATTUUAG GAGGUCUCUCUUUGGTT) and Mad2 (GGACUCACCUUGCUUACAATTU UGUAAGCAAGG U G A GUCCTT) (Gene Pharma) or siRNA-negative controls were microinjected (5 μM) into fully grown immature oocytes with an Eppendorf microinjection instrument (Hamburg) and allowed to incubate for 30 min. Oocytes were arrested in the GV stage in MI6 medium (Sigma-Aldrich) supplemented with 2.5 μM milrinone for 20-24 h. The oocytes were then thoroughly washed with DPBS to resume meiosis.

| Investigation of mitochondrial distributions
Mitochondrial distributions were evaluated using the mitochondrial reactive dye Mito-tracker (Green) (Beyotime Institute of Biotechnology). Oocytes were placed in maturation medium with 5 μM Mito-tracker (Green) for 20 min and thoroughly washed three times with DPBS. A confocal laser scanning microscope (Nikon A1R) was then used to investigate the oocytes, and an NIS-Elements AR (Nikon Instruments) was used to quantify mitochondria.

| Quantification of mitochondrial membrane potentials
A mitochondrial membrane potential assay kit (JC-1 dye, Beyotime Institute of Biotechnology) was used to measure mitochondrial membrane potentials (Δφm). Oocytes were stained with a working solution containing 10 μM JC-1 at 37.0°C in a 5% CO 2 atmosphere for 20 min, after which they were washed with washing buffer (DPBS) to remove surface fluorescence, followed by observation with a fluorescence microscope (Olympus IX73). Red fluorescence corresponded to activated mitochondria (J-aggregates), while green fluorescence corresponded to less activated mitochondria (J-monomers), and the ratio is given as the Δφm value.

| ATP content assays
The ATP content in each oocyte was measured with an Enhanced ATP Assay Kit, S0027 (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Different ATP standards were prepared, ranging from 0 to 40 pol 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 min 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 to sit at room temperature for 3-5 min.
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).

| Immunofluorescence
Mouse MI or MII oocytes were fixed in 4%(w/v) paraformaldehyde for 40 min at room temperature and washed three times (10 min 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 h and washed three times The oocytes were then expanded on glass slides and examined with confocal laser-scanning microscopy (FLUOVIEW FV1000, Olympus) using the FLUOVIEW Viewer (Olympus). The excitation lasers were set at 488 nm, and emission channels of 520 nm were used for green fluorescence detection.

| RNA sequencing
We performed expression profiling on pools of 30 denuded GV oocytes isolated per group. RNA was isolated using the RNeasy Micro Kit (Qiagen). cDNA was generated and amplified from 1. Demultiplexing and fast-q generation were performed with bcl2fastq (bcl2fastq-1.8.3).

| RNA extraction, reverse transcription and quantitative PCR (Q-PCR)
Total RNA was extracted from 40 GV, MI or MII oocytes using a RNeasy micro-RNA isolation kit (Qiagen) following the manufacturer's instructions. Samples were treated with DNase I, and then Transcript-Uni Cell was used for cDNA Synthesis. A Q-PCR supermix was used for the assays (Trans Gen Biotech). RNA concentrations were measured using a Nanodrop 2000 Spectrophotometer (Biolab, Scoresby) at a wavelength of 260 nm. Samples for subsequent analyses were only used if their 260:280 nm absorbance ratios were >1.8. Primers for the published reference RNA sequences for real-time Q-PCR and RT-PCR are listed in Table 1.Q-PCR and RT-PCR assays were performed with an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems), respectively. Three replicates were conducted for all assays. The relative expression of genes was calculated with the comparative threshold cycle (CT) method as 2 -ΔΔCT . The primers used for the amplification assays are shown in Table 2.

| Statistical analyses
All experiments were repeated at least three times. Data are presented as means ± SEM, unless otherwise stated. Statistical comparisons were made with Student's t tests or one-way ANOVA tests, where appropriate. A p < 0.05 was considered statistically significant.

| Mitochondrial Ca 2+ and ATP levels during oocyte maturation
Mitochondrial Ca 2+ and ATP levels were first evaluated during oocyte meiotic maturation by immunofluorescent staining and confocal microscopy. Quantitative analysis of mitochondrial Ca 2+ and ATP contents indicated that changes in ATP content were consistent with mitochondrial calcium changes ( Figure 1A-C). Thus, mitochondrial calcium influxes may play a key role during oocyte maturation. We consequently attempted to evaluate the upstream and downstream relationships between mitochondrial calcium and ATP during oocyte maturation. We postulated that mitochondrial Ca 2+ had a direct effect on ATP production, based on our previous results. 18 To address this possibility, we treated fully grown oocytes at the GV stage with different concentrations of Ru360 or oligomycin, which are inhibitors of mitochondrial Ca 2+ influx or ATP synthesis. The low levels of mitochondrial Ca 2+ resulted in lower ATP contents ( Figure 1D-E), but low ATP contents had no effect on mitochondrial Ca 2+ levels, suggesting that mitochondrial Ca 2+ downregulated ATP production in mouse oocytes.

| MITOCHONDRIAL CALCIUM UNIPORTERS is important for meiotic progression in mouse oocytes
To further investigate MCU function, a specific siRNA microinjection procedure was used with fully grown oocytes at the GV stage.  knockdowns, 6.7% vs.35.7%, Figure 3A,B) and MII (controls vs. knockdowns, 12.1% vs. 51.4%, Figure 3C,D). Additionally, Ru360, a specific inhibitor of MCU, was used to validate the siRNA results.
As expected, result showed that the effect of Ru360 on meiotic progress and spindle assembly of oocytes was similar to knockdown of MCU with siRNA ( Figure S2A-G). Together, these findings indicate that MCU is required for orderly oocyte meiotic maturation and that MCU knockdown oocytes are unable to properly assemble spindles during meiosis.

| Mitochondrial Calcium uniporters is important for maintaining mitochondrial calcium homeostasis in mouse oocytes
Given that MCU had an important role in maintaining mitochon-

| Recognition of key effectors of si-Mitochondrial Calcium Uniporters oocytes by singlecell transcriptome analysis
To delineate the genes and pathways affected in knockdown treatment on the quality of oocytes, we performed single-cell transcrip- As shown in Figure 5A,B, we used gene ontology (GO) analysis, showing that misregulated genes revealed overrepresentation of several biological processes. Especially in downregulated Go analysis, we found cytokine-metabolic process changed evidently, which might decline energy synthesis in oocytes. Additionally, we noticed that reproduction development progress, actin-cytoskeleton organization and mitochondrial development progress also had negative regulation, which indicated that some pivotal factor in oocytes quality had been damaged. Hence, all of these pathways or biological processes are highly related to energy metabolism, oxidative stress mitochondrial function, which prompts us to focus on mitochondria and energy sense reaction in si-MCU oocytes.

| Mitochondrial Calcium Uniporters is required for the maintenance of mitochondrial function and ATP content during oocyte meiosis
Since abnormal mitochondrial calcium signalling can significantly

| Mitochondrial Calcium Uniporters knockdown activates the AMPK signal in mouse oocytes
The effects of MCU defects on meiotic maturation and mitochondrial function led us to evaluate the potential mechanisms that would explain these phenotypes. From GO analysis, we noticed that some upregulated genes concentrated in AMPK signal ( Figure 5B). Since a lower AMPK activity is activated by low ATP levels ( Figure 7D,E), we expressed p-AMPK in GV and MII oocytes by antibodies with immunofluorescence labels. Consistent with our hypothesis, immunofluorescence microscopy indicated that p-AMPK exhibited significantly upregulated expression in oocytes after MCU depletion ( Figure 7D,E). Si-MCU oocytes at the GV stage were then treated with different concentrations of a distinct inhibitor of AMPK signal, Compound C. The inhibitor ameliorated meiotic maturation defects in MCU defection oocytes (Table 1). Collectively, these results suggest that MCU knockdown activates AMPK signalling in mouse oocytes and abnormally activated AMPK has universally adverse effects on meiosis progress ( Figure 8).

| DISCUSS ION
Fully grown oocytes are rich with many kinds of maternal factors that are indispensable for meiotic maturation, fertilization and early embryonic development. 19,20 Furthermore, mitochondrially produced ATP acts as the basic energy source for many biological processes, since normal mitochondrial function plays a key role in maintaining ATP levels. 15

ACK N OWLED G EM ENTS
Not applicable

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

O RCI D
Zhuan Qingrui https://orcid.org/0000-0003-2797-0472 Hou Yunpeng https://orcid.org/0000-0002-6807-8336 F I G U R E 8 Model for MCU-mediated ATP synthesis and abnormal AMPK activity in meiosis. When si-MCU occurs in oocytes due to impaired mitochondrial function, cytosolic ATP levels decline. The energy sensor AMPK is activated and phosphorylated in response to the increased energetic stress. Excessive activation of AMPK results in adverse effects on the resumption of meiosis. In addition, microtubule dynamics and tension establishment cannot efficiently be achieved, leading to decreased meiotic progression. These observations all implicate MCU as being critical for meiotic progression