Regulation of [Ca2+]i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

Oocyte activation inefficiency is one of the reasons for female infertility and Ca2+ functions play a critical role in the regulation of oocyte activation. We used various inhibitors of Ca2+ channels located on the membrane, including sarcoplasmic/ endoplasmic reticulum Ca2+ATPases (SERCAs, the main Ca2+ pumps which decrease the intracellular Ca2+ level by refilling Ca2+ into the sarcoplasmic reticulum), transient receptor potential (TRP) ion channel subfamily member 7 (TRPM7, a Ca2+/Mg2+-permeable non-selective cation channel), T-type Ca2+ channels and calcium channel Orai1, to investigate their roles in [Ca2+]i oscillation patterns and mitochondrial membrane potential during oocyte activation by real-time recording. Our results showed that SERCAs, TRPM7 and T-type Ca2+ channels were important for initiation and maintenance of [Ca2+]i oscillations, which was required for mitochondrial membrane potential elevation during oocyte activation, as well as oocyte cytoskeleton stability and subsequent embryo development. Increasing the knowledge of calcium transport may provide a theoretical basis for improving oocyte activation in human assisted reproduction clinics.


Introduction
According to reports by the World Health Organization in 2016, at least one of ten couples in developed countries cannot have children within 5 years of marriage, half of which are due to female infertility [1]. Oocyte activation inefficiency is a major problem causing female infertility [2]. Oocytes are arrested at metaphase of the second meiosis until fertilization takes place. Directly following sperm penetration, oocyte activation begins with a series of crucial steps triggered by periodical repetitive increases and decreases of intracellular Ca 2+ ([Ca 2+ ] i ) concentrations known as [Ca 2+ ] i oscillations [3,4]. Although [Ca 2+ ] i oscillations are required for oocyte activation [5], very little is known about which Ca 2+ transporter participates in producing [Ca 2+ ] i oscillations during oocyte activation.
The importance of Ca 2+ functions in the regulation of oocyte activation is increasingly being recognized [6]. There are many important channel proteins involved in Ca 2+ transport in oocytes. Ca 2+ transporters on the plasma membrane can be controlled by voltage, ligand, second messengers, Ca 2+ concentration, or other interactions [7]. Ca 2+ influx into the cytoplasm is mediated by a diverse population of Ca 2+ transporters exhibiting significant diversities in their gating and activation mechanisms. Once the oocyte is activated, the [Ca 2+ ] i oscillations are produced by simultaneous intracellular Ca 2+ storage release and the extracellular Ca 2+ influx [8]. Hence, it is important to elucidate the molecular mechanisms of [Ca 2+ ] i oscillations at activation. According to our previous research, mitochondria, main organelles for energy production in the cell, are involved in producing [Ca 2+ ] i oscillations during oocyte activation [9]. Since Ca 2+ regulates mitochondria activity in cardiomyocytes [10], we thus believe that [Ca 2+ ] i oscillations in the cytoplasm will have similar effects in oocytes. At present, the transporters involved in the regulation of [Ca 2+ ] i oscillations and mitochondria activity have not been fully determined.
In this study, specific inhibitors for different Ca 2+ transporters were introduced to detect the [Ca 2+ ] i oscillation patterns and mitochondria membrane potential dynamic changes, in order to evaluate the role of Ca 2+ transporters in oocyte activation. Studying the function and regulatory mechanisms of these transporters will have significant importance for both understanding oocyte activation mechanisms and for improving clinical assisted reproductive technologies (ART).

Ethics statement
Female ICR mice at 8 weeks old were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were handled in accordance with the institutional animal care policies of the Institute of Zoology, Chinese Academy of Sciences. Mice were maintained under a 12-h light and 12-h darkness cycle in a specific pathogen-free stage at the Central Animal Laboratory of the Institute of Zoology, Chinese Academy of Sciences. The Laboratory Animal Care and Use Committee of the Institute of Zoology approved this study (The ethics committee approval number: SYXK 2018-0021).

In vitro transcription of cRNA
The cytoplasmic spindle formation and F-actin meshwork were investigated by live imaging of F-actin with mRNA encoding b5-tubulin-GFP [11,12] and UtrCH-eGFP [11,13] as previously described. Templates of in vitro transcription from constructed plasmids were obtained by PCR with F and R primers. PCR products were diluted in RNAse-free water. cRNA transcripts were synthesized in vitro with T7 RNA polymerase mMESSAGE mMACHINE T7 kit (Ambion, Life. Co, Calsbad, CA, USA). Poly(A) tail was added to the sequence end by polymerase tailing kit (PAP5104, Lucigen, Beijing, China). The RNA solutions were then stored at − 80°C in a final concentration of 400 μg/mL until further use. Approximately 50 pl of RNA solution was injected into each GV oocyte.

Oocyte collection and partheno-activation
Female mice were injected with 10 IU pregnant mare serum gonadotropin (PMSG, Ningbo Hormone Product Co. Ltd., Cixi, China). After 44 to 48 h, the GV oocytes were collected by ovarian mincing. Female mice were injected with 10 IU PMSG followed 44 to 48 h later by injection of 10 IU human chorionic gonadotropin (hCG, Ningbo Hormone Product Co. Ltd., Cixi, China). After 15 h of hCG, ovulated MII oocytes were collected and denuded in 1 mg/ml hyaluronidase. [Ca 2+ ]i oscillations were induced by 10 mM strontium chloride (SrCl 2 , 10,025-70-4, Sangon Biotech, Shanghai, China) in Ca 2+ -free CZB culture medium containing various Ca 2+ channel modulators for as long as 4 h. After partheno-activation, embryos were moved into KSOM (MR-106, Merck Millipore, USA) for further culture, and pronuclear formation and cleavage were observed.

The GV oocyte microinjection
The M2 medium and M16 medium containing 2.5 μM milrinone were prepared and warmed to 37°C. Milrinone is a phosphodiesterase inhibitor that maintains meiotic arrest once oocytes are removed from the follicles [18]. Micro-drops each containing 20 μl M16 medium with milrinone were prepared in a dish and overlaid with mineral oil. Injection pipettes were made by pulling borosilicate-glass capillary with filament in a mechanical puller. We used a Flaming-Brown micropipette puller (Model P-97) with the following settings: P = 540, Heat = 300, Pull = 130, Vel = 100, Time = 150. Microinjection platform was prepared by placing a 10 μl micro-drop of M2 medium with milrinone on a chamber slide, and the drop was covered with mineral oil and placed on the microscope stage. Injection and holding pipettes were placed into the drop of M2 medium with milrinone. cRNA microinjection of GV oocytes was performed with Narishige micromanipulators (Narishige Inc., Sea Cliff, NY) under a Nikon TE 200 (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) and finished within 30 min. After cRNA injection, the oocytes were arrested at the GV stage in M16 medium containing 2.5 μM milrinone for 4 h. Then the oocytes were transferred to M16 medium and cultured under mineral oil at 37°C, in an atmosphere of 5% CO 2 in air for 14 h in vitro maturation.

Real-time recording of [Ca 2+ ]i changes
Oocyte [Ca 2+ ] i oscillations were detected using 2 μg/mL Fluo-4 AM (488 nm excitation, 525 nm emission) in the partheno-activation solution. Real-time images of [Ca 2+ ] i changes after partheno-activation of oocytes which were co-incubated with various Ca 2+ channel modulators were obtained using a time-lapse confocal laser microscope (UltraVIEW-VoX; PerkinElmer, MA, USA) and recorded at 2 frames per minute. [Ca 2+ ] i intensity was detected using an argon laser. Volocity Software was used to analyze fluorescence intensity.

Real-time recording of oocyte mitochondrial potential
In our study, we set out to confirm the [Ca 2+ ] i oscillations regulatory function in mitochondrial activity during oocyte activation. Oocytes were denuded and pre-incubated in M2 culture medium (M7167, Sigma-Aldrich, USA) with 2 μg/mL JC-1 (C2005, Beyotime, Beijing, China), a cell permeable voltage-sensitive fluorescent mitochondrial dye, for 15 min. In order to reduce the toxicity of JC-1 for long-term observation using laser confocal microscope, we reduced the concentration of JC-1 to 0.5 μg/ml in the activating solution. Briefly, the lower potential mitochondria were imaged as JC-1 monomer emitting in green (488 nm excitation, 561 nm emission), and energized high potential mitochondria were imaged in aggregated JC-1 that emits in red (488 nm excitation, 561 nm emission) as fluorescence transporters. Oocyte mitochondrial potential dynamic changes after treatment with various Ca 2+ channel modulators were recorded after partheno-activation using a time-lapse confocal laser microscope (UltraVIEW-VoX; PerkinElmer, MA, USA) at identical magnification and gain settings throughout the experiments. A software Volocity was used to analyze fluorescence intensity.

Statistical analysis
All experiments were conducted at least three times. At least 40 oocytes were collected and examined in each experiment and experiments were repeated more than three times. More than a total of 120 oocytes were examined in each group. We presented information of samples with Means and Standard Deviations (SD). Results were analyzed by SPSS 19.0. The significance of differences among groups was analyzed by the Chi-square test, and p values less than 0.05 were considered statistically significant.

Inhibitors of Ca 2+ channels affect the efficiency of oocyte activation
We firstly examined the dose-dependent effects of inhibitors of SERCAs, TRPM7, T-type Ca 2+ channels and Orai1 on the efficiency of SrCl 2 -induced oocyte parthenoactivation and found a suitable concentration. The percentages of survival oocytes showed clear cell membrane and high cytoplasmic refraction after activation, pronuclear formation and 2-cell cleaved embryos as shown in Table 1. We applied Thapsigargin (Tha) with gradient concentrations of 0.5, 1, and 10 μM. When its concentration rose to 10 μM, [Ca2+]i would rise slowly, and oocytes died during the activation process. NS-8593 was applied with gradient concentrations of 0.1, 1, and  (Fig. 1a). We found that mitochondria were activated from the cortex to the internal area of the oocyte and finally aggregated around the chromosomes (Fig. 1b). In order to analyze changes in oocyte mitochondrial membrane potential, a relative fluorescence intensity analysis and a high membrane potential/low membrane potential ratio were introduced. Ordinate was marked as Relative Fluorescence Intensity (relative to the fluorescence intensity of the start point).
The overall membrane potential showed two elevated peaks and then returned to a basal level (Fig. 1c, d).
Oocyte cytoskeleton (spindle in Fig. 1e, and f-actin in Fig. 1f) showed a clear dynamic distribution during activation.

Effect of SERCAs inhibitor Thapsigargin on oocyte activation
Endoplasmic reticulum (ER) is a major Ca 2+ storage component in the cytoplasm. SERCAs are the main transporters for cytoplasmic Ca 2+ refilling into the ER. We wanted to observe changes in [Ca 2+ ] i oscillations and mitochondrial activity during oocyte activation after the Ca 2+ refill was blocked by SERCAs inhibitor Thapsigargin (Tha) (Fig. 2). The oocyte survival rate was 66.2% when treating with 1 μM Tha (Table. 1), by which concentration [Ca 2+ ] i oscillations (Fig. 2a) and pronuclear formation were sufficiently suppressed. Mitochondrial membrane potential continued to decrease under Ca 2+ refilling inhibition by 1 μM Tha (Fig. 2b). At a concentration of 10 μM Tha completely inhibited Ca 2+ refill into ER, and oocyte died during activation (Fig. 2a).
Ordinate was marked as Relative Fluorescence Intensity (relative to the fluorescence intensity of the start point). Spindle (Fig. 2d) and F-actin (Fig. 2e) showed dispersion when 1 μM Tha caused oocyte death. Thus, Ca 2+ refilling into ER through SERCAs plays an important role in [Ca 2+ ] i oscillations and mitochondrial activation during oocyte activation.

Effect of TRPM7 inhibitor NS-8593 on oocyte activation
TRPM7 is highly expressed in the GV and MII oocytes, mainly distributed on the plasma membrane. We inhibited TRPM7 activity by its specific inhibitor NS-8593 to study its role in oocyte activation (Fig. 3). The [Ca 2+ ] i oscillations (Fig. 3a) and pronuclear formation were sufficiently suppressed when 1 μM NS-8593 was used to inhibit the transport of Ca 2+ through TRPM7. The dynamic mitochondrial membrane potential of oocytes was observed after treatment with 1 μM NS-8593 (Fig. 3b). At this concentration, mitochondrial membrane potential continuously decreased. Depolarization of mitochondria was found in the case of long-term inhibition (Fig. 3b). When treating with 5 μM NS-8593, [Ca 2+ ] i slowly rose and finally induced oocyte death. Ordinate was marked as Relative Fluorescence Intensity (relative to the fluorescence intensity of the start point). Spindle (Fig. 3d) and F-actin (Fig. 3e) (Fig. 4). The mitochondrial membrane potential was observed in the 5 μM group. We found that the mitochondrial membrane potential continued to decrease after inhibiting the transport of Ca 2+ through T-type Ca 2+ channels. When the concentration was increased to 10 μM, Ca 2+ slowly increased, which induced rapid death of most oocytes. Ordinate was marked as Relative Fluorescence Intensity (relative to the fluorescence intensity of the start point). Spindle (Fig. 3d) and F-actin (Fig. 3e) showed dispersion when 5 μM Mibefradil caused oocyte death. Based on these results it is concluded that the transport of Ca 2+ through T-type Ca 2+ channels is important for [Ca 2+ ] i oscillations and mitochondrial activity in oocyte activation and survival.

Effect of Orai1 inhibitor GSK-7975A on oocyte activation
Orai1 is distributed on the plasma membrane. We studied the role of Orai1 in oocyte activation by treating oocytes with the specific inhibitor GSK-7975A. The mitochondrial membrane potential was observed at 1 mM, which showed irregular changes compared to the Ctrl group (Fig. 5).
Most oocytes survived until 4 h of activation, but none of them formed a pronucleus due to cytotoxicity. Ordinate was marked as Relative Fluorescence Intensity (relative to the fluorescence intensity of the start point). It was not confirmed whether this [Ca 2+ ] i oscillation pattern and mitochondria membrane potential changes were caused by complete inhibition of Orai1 or by cytotoxicity induced by the high concentration of 1 mM GSK-7975A. Oocytes spindle (Fig. 3d) showed a chromosome segregation and F-actin false distribution when 1 mM GSK-7975A caused oocyte fragmentation. The role of Orai1 in the activation of oocytes requires additional evidence to confirm.

Effects of Ca 2+ channel inhibitors on long-lasting [Ca 2+ ] i oscillations
The mechanisms of initiation and maintenance of [Ca 2+ ] i oscillations are believed to be different. However, it is unclear which transporters participate in the initiation or maintenance. The effects of several transporters on the maintenance of [Ca 2+ ] i oscillations were investigated in our study (Fig. 6). By using time-lapse confocal  (Fig. 6a). The same concentration was applied as in the mitochondrial membrane potential observation study mentioned above. Ruthenium Red (MACKLIN, Beijing, China) completely inhibited oocyte activation as a non-specific cation channel inhibitor. It was found that the addition of Ruthenium

Discussion
Obese, diabetic and aging women typically suffer from abnormal body metabolism such as hypertension, hyperglycemia and hyperlipidemia [19][20][21][22][23][24], causing long-term stress in oocytes [25], which severely damages the quality of oocytes, thereby leading to lower pregnancy rates [26]. Insufficient oocyte activation and mitochondrial damage were considered to be major causes for embryo developmental disorders [27,28]. Ca 2+ is one of the major signal molecules that regulate various cell functions including cell cycle progression, arrest and apoptosis. Oocyte activation induces a continuous series of oocyte intracellular Ca 2+ concentration ([Ca 2+ ] i ) increases and decreases known as [Ca 2+ ] i oscillations, which encode oocyte activation events, including release from the MII arrest, pronuclear formation and the transition to embryo development [29]. Release of Ca 2+ from internal stores and Ca 2+ influx from the extracellular matrix induce moderate increases in [Ca 2+ ] i levels. The increase in [Ca 2+ ] i generally lasts about 2 min. As Ca 2+ refills back into the ER or causes efflux from the cytoplasm to prepare for the next peak of oscillation, the elevated [Ca 2+ ] i will return to baseline levels, resulting in an average of a ten to twenty minutes The significance of differences between groups was analyzed by the Chi-square test and p < 0.05 (*) was considered statistically significant. (X) indicates data unavailable resting interval. [Ca 2+ ] i oscillations will last 5-6 h until pronuclear formation takes place [9]. The repeated elevation and recovery of Ca 2+ signaling is tightly regulated, and the strictly ordered Ca 2+ signal will coordinate the interaction of various organelles in the oocyte for its activation. Ca 2+ transporters and regulators could become potential therapy targets for in vitro fertilization failure.
During cell activation, Ca 2+ shuttles through the cell, and the transporter is likely to be located near IP3R and downstream organelles [30]; the cell membrane, lysosomes, the nucleus, vesicles and mitochondria may be targets of Ca 2+ release. Endoplasmic reticulum (ER) is a main intracellular Ca 2+ store, where the Ca 2+ concentration increases to 300 or even 1000 μM [31]. The pathway for Ca 2+ efflux from the ER into the cytoplasm has not yet been well identified. SOCE or Ca 2+ releaseactivated Ca 2+ channels (CRAC), were first described in immune cells where they have been shown to be critical for their function. Accordingly, defects in SOCE in humans are associated with severe immune-deficiencies [32]. In oocytes, the predominant [Ca 2+ ] i increase pathway appears to be achieved through store-operated Ca 2+ entry (SOCE) processes. Ca 2+ enters the cytosol from the endoplasmic reticulum (ER), which in turn opens one of the ER channels, sarcoplasmic reticulum/ER Ca-ATPase (SERCAs), to transport Ca 2+ back to the ER [33]. Total cellular Ca 2+ was estimated by the addition of 10 μM Thapsigargin (Tha), a SERCAs inhibitor, which induced complete release of Ca 2+ from ER [34]. When ER Ca 2+ stores had been significantly depleted by Tha, sperm no longer triggered [Ca 2+ ] i oscillations [35]. In our study, we used three gradient concentrations of 0.5, 1 and 10 μM of Tha. Ten μM Tha kept oocytes at a higher [Ca 2+ ] i and all oocytes died before the end of the activation process (Fig. 2). However, when oocyte [Ca 2+ ] i oscillations were suppressed by 1 μM Tha, more than half of the oocytes survived more than 4 h. Under such moderate concentration, the effects of SERCAs on mitochondrial activity can be observed for a long term. Oocyte mitochondrial membrane potential continued to decrease under Ca 2+ refilling inhibition with 1 μM Tha incubation. Not only [Ca 2+ ] i oscillations but also mitochondrial activity were suppressed by Tha that induced blocking of ER Ca 2+ refilling. Finally, oocytes cannot be activated under SERCAs inhibition.
Recently, a member of the TRP channels family, TRPM7, was found to be expressed in mouse GV, MII oocytes and 2-cell embryos [36]. TRPM7 belongs to the subfamily of melastatin and exhibits a ubiquitous tissue distribution. Trpm7 knock-out caused E14.5 embryonic lethality [37]. Using inhibitor NS-8593 suppression of the transporter hours after activation reduced progression to the blastocyst stage [36]. Oocytes treated with 10 μM NS-8593 and fertilized in vitro display impaired Ca 2+ oscillations [15]. We applied NS-8593 with gradient concentrations of 0.1, 1, and 5 μM (Tab. 1 and Fig. 3a). After treating with 5 μM NS-8593, [Ca 2+ ] i slowly rose, which finally induced oocyte death. Treatment with 1 μM NS-8593 kept the survival rate at 37.5% (Fig. 3), while the [Ca 2+ ] i oscillations and pronuclear formation were sufficiently suppressed. Under this moderate concentration of 1 μM NS-8593, the effects of TRPM7 on mitochondrial activity can be observed for a long time. Mitochondrial activity did not exhibit the same activation state as the Ctrl in the case of TRPM7 inhibition with NS-8593. Mitochondrial membrane potential continuously decreased when inhibiting the transport of Ca 2+ through TRPM7 with 1 μM NS-8593 (Fig. 3). The TRPM7 on the cell membrane of oocytes has a significant effect on the [Ca 2+ ] i oscillation patterns in oocytes, and [Ca 2+ ] i oscillations achieved through TRPM7 is important for mitochondrial activity and oocyte activation.
Mibefradil is a T-type channel inhibitor. Mibefradil was developed as a cardiovascular hypertension and angina remedy [38]. Mibefradil has been repurposed as an anti-cancer drug [39]. However, its underlying mechanisms are still unclear. The mechanism of the anticancer therapy is thought to be via the blockage of Ca 2+ influx through T-type channels [40]. Mibefradil blocks all three subtypes of T-type channels [41], including Cav3.1, Cav3.2, and Cav3.3, with an IC50 (semi-lethal concentration) of 5.8-7.2 μM. In our study, inhibition of Ca 2+ release by high concentrations of Mibefradil impaired intracellular Ca 2+ dynamics and thus affected cell viability. We applied Mibefradil at three gradient concentrations of 0.5, 5, and 10 μM (Tab. 1 and Fig. 4). In order to conduct long term observations, a moderate concentration of 5 μM NS-8593 was selected to study the effects of T-type channels on mitochondrial activity. The mitochondrial membrane potential continued to decrease after inhibiting the transport of Ca 2+ through T-type Ca 2+ channels with 5 μM Mibefradil. When the concentration reached 10 μM, Ca 2+ rapidly increased, which induced rapid death of most oocytes. Thus, transport of Ca 2+ through T-type Ca 2+ channels is important for mitochondrial activity and oocyte activation.
Orais represents transmembrane proteins that form highly Ca 2+ -selective channels [42]. Orais has three family members, ORAI1, 2 and 3 [43]. Loss-of-function mutation of ORAI1 caused immune deficiency [44] and dysfunction of thrombus formation [45]. SOCE is also mediated through the ORAI channels at the outer membrane. 10 μM GSK-7975A has been reported to induce maximal inhibition of Ca 2+ influx in Jurkat T-cells [17]. We applied GSK-7975A at gradient concentrations of 10, 100 μM, and 1 mM. We found that oocytes could not be activated when GSK-7975A below 1 mM was used to effectively inhibit Ca 2+ influx. Mitochondrial dynamic membrane potential showed irregular changes compared to the Ctrl group (Fig. 5). Most of the oocytes survived up to 4 h post activation even at a concentration as high as 1 mM, but none of them formed a pronucleus. Female mice were fertile after knocking out ORAI1 [15]. It is not yet clear whether this [Ca 2+ ] i oscillation pattern and membrane potential changes were caused by complete inhibition of Orai1 or by cytotoxicity induced by the high concentration of 1 mM. The role of Orai1 in the activation of oocytes requires additional evidence to confirm.
The mechanisms of initiation and maintenance of [Ca 2+ ] i oscillations are different [46]. However, it is unclear which transporters participate in the initiation or maintenance of [Ca 2+ ] i oscillations. The effects of several transporters on the maintenance of [Ca 2+ ] i oscillations were investigated in our study (Fig. 6). We found that the addition of Ruthenium Red, Thapsigargin and GSK-7975A all inhibited the maintenance of [Ca 2+ ] i oscillations. After addition of Mibefradil and NS-8593, the intracellular Ca 2+ increased continuously, and the cell death rate was higher than in the Ctrl and other groups. All these results suggest that three Ca 2+ transporters, SERCAs, TRPM7 and T-type Ca 2+ channels are involved in both the initiation and maintenance of [Ca 2+ ] i oscillations. Interestingly, the addition of Ruthenium Red and other inhibitors into culture of oocytes which had initiated [Ca 2+ ] i oscillations did not significantly influence oocyte activation (Fig. 6b), suggesting that oocyte activation required only a small amount of [Ca 2+ ] i oscillations.
In summary, we applied ER-associated Ca 2+ transporter SERCAs inhibitor Thapsigargin, TRPM7 inhibitor NS-8593, T-type Ca 2+ channels inhibitor Mibefradil, and Orai1 inhibitor GSK-7975A to understand the regulation of [Ca 2+ ] i oscillations and mitochondrial activity during oocyte activation, and showed that inhibition of SERC As, TRPM7 and T-type Ca 2+ channels caused disturbances in Ca 2+ signaling, mitochondrial activity and subsequent oocyte activation and embryonic development. Once Ca 2+ transport is abnormal in oocytes, whether it is at the beginning or halfway, will cause disorders of mitochondrial metabolism, activation of subsequent developmental consequences, finally leading to infertility.