Obese, diabetic and aging women typically suffer from abnormal body metabolism such as hypertension, hyperglycemia and hyperlipidemia [11–16], causing long-term stress in oocytes [17], which severely damages the quality of oocytes, thereby leading to lower pregnancy rate [18]. Insufficient oocyte activation and mitochondrial damage were considered to be major causes for embryo developmental disorders[19, 20].
Ca2+ 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 Ca2+ concentration ([Ca2+]i) increases and decreases known as [Ca2+]i oscillations, which encode oocyte activation events, including liberation from the MII arrest, pronucleus formation and the transition to embryo development [21]. Release of Ca2+ from internal stores and Ca2+ influx from the extracellular matrix induce moderate increases in [Ca2+]i levels. The increase of [Ca2+]i generally lasts about 2 minutes. As Ca2+ refills back to the ER or efflux from the cytoplasm to prepare for the next peak of oscillation, the elevated [Ca2+]i will return to baseline levels, resulting in an average of a ten to twenty minutes resting interval. [Ca2+]i oscillations will last 5–6 hours until pronuclear formation [9]. The repeated elevation and recovery of Ca2+ signaling is tightly regulated, and the strictly ordered Ca2+ signal will coordinate the interaction of various organelles in the oocyte for its activation. Ca2+ transporters and regulators could become potential therapy targets for infertile women especially for in vitro fertilization failure.
During activation, Ca2+ shuttles through the cell, and the transporter is likely to be located near IP3R and downstream organelles [22]; the cell membrane, lysosomes, the nucleus, vesicles and mitochondria may be targets of Ca2+ release. Endoplasmic reticulum (ER) is a main intracellular Ca2+ store, where the Ca2+ concentration increases to 300 or even 1,000 µM [23]. The pathway for Ca2+ efflux from the ER into the cytoplasm has not yet been well identified. SOCE or Ca2+ release-activated Ca2+ 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 [24]. In oocytes, the predominant [Ca2+]i increase pathway appears to be achieved through store-operated Ca2+ entry (SOCE) processes. Ca2+ enters the cytosol from the endoplasmic reticulum (ER), which in turn opens one of ER channel, sarcoplasmic reticulum/ER Ca-ATPase (SERCAs), to transport Ca2+ back to ER [25]. Total cellular Ca2+ was estimated by the addition of 10 µM Thapsigargin (Tha), a SERCAs inhibitor, which induced complete release of Ca2+ from ER [26]. When ER Ca2+ stores had been significantly depleted by Tha, sperm no longer triggered [Ca2+]i oscillations [27]. 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 [Ca2+]i and all oocytes died before the end of the activation process (Fig. 2). However, when oocyte [Ca2+]i oscillations were suppressed by 1 µM Tha, more than half of the oocytes survived more than 4 hours. Under such moderate concentration, the effects of SERCAs on mitochondrial activity can be observed for a long term. Oocyte mitochondria membrane potential continued to decrease under Ca2+ refilling inhibition with 1 µM Tha incubation. Not only [Ca2+]i oscillations but also mitochondria activity were suppressed by Tha that induced blocking of ER Ca2+ refilling. Finally, oocytes cannot be activited 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 [28]. TRPM7 belongs to the subfamily of melastatin and exhibits a ubiquitous tissue distribution. Trpm7 knock-out caused E14.5 embryonic lethality [29]. Using inhibitor NS-8593 suppression of the transporter hours after activation reduced progression to the blastocyst stage [28]. Oocytes treated with 10 µM NS-8593 and fertilized in vitro display impaired Ca2+ oscillations [30]. We applied NS-8593 with gradient concentrations of 0.1, 1, and 5 µM (Table 1 and Fig. 3A). After treating with 5 µM NS-8593, [Ca2+]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 [Ca2+]i oscillations and pronuclear formation were sufficiently suppressed. Under such moderate concentration of 1 µM NS-8593, the effects of TRPM7 on mitochondrial activity can be observed for a long term. Mitochondrial activity did not exhibit the same activation state as the Ctrl in the case of TRPM7 inhibition with NS-8593. Mitochondrial membrane potential continually decreased when inhibiting the transport of Ca2+ through TRPM7 with 1 µM NS-8593 (Fig. 3). The TRPM7 on the cell membrane of oocytes has a significant effect on the [Ca2+]i oscillation patterns in oocytes, and [Ca2+]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 [31]. Mibefradil has been repurposed as an anti-cancer drug [32]. However, its underlying mechanisms are still unclear. The mechanism of the anti-cancer therapy is thought to be via the blockage of Ca2+ influx through T-type channels. Mibefradil blocks all three subtypes of T-type channels, 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 Ca2+ release by high concentrations of Mibefradil impaired intracellular Ca2+ dynamics and thus affected cell viability. We applied Mibefradil at three gradient concentrations of 0.5, 5, and 10 µM (Table 1 and Fig. 4). In order to observe for a long term, 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 Ca2+ through T-type Ca2+ channels with 5 µM Mibefradil. When the concentration reached 10 µM, Ca2+ rapidly increased, which induced rapid death in most oocytes. Thus, transport of Ca2+ through T-type Ca2+ channels is important for mitochondrial activity and oocyte activation.
Orais are 4 transmembrane proteins that form highly Ca2+-selective channels [33]. Orais has three family members, ORAI1, 2 and 3 [34]. Loss-of-function mutation of ORAI1 caused immune deficiency [35] and dysfunction of thrombus formation [36]. SOCE is also mediated through the ORAI channels at the outer membrane. 10 µM GSK-7975A has been reported to induce maximal inhibition of Ca2+ influx in Jurkat T-cells [37]. 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 Ca2+ influx. Mitochondrial dynamic membrane potential showed irregular changes compared to the Ctrl group (Fig. 5). Most of the oocytes survived up to 4 hours 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 [30]. It is not yet clear whether this [Ca2+]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 [Ca2+]i oscillations are different [38]. However, it is unclear which transporters participate in the initiation or maintenance of [Ca2+]i oscillations. The effects of several transporters on the maintenance of [Ca2+]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 [Ca2+]i oscillations. After addition of Mibefradil and NS-8593, the intracellular Ca2+ increased continuously, and the cell death rate was higher than in the ctrl and other groups. All these results suggest that three Ca2+ transporters, SERCAs, TRPM7 and T-type Ca2+ channels are invilved in both the initiation and maintenanceof [Ca2+]i oscillations. Interestingly, the addition of Ruthenium Red and other inhibitors into culture of oocytes which had initiated [Ca2+]i oscillations did not significantly influence oocyte activation (Fig. 6B), suggesting that oocyte activation required only a small amount of [Ca2+]i oscillations.
In summary, we applied ER-associated Ca2+ transporter SERCAs inhibitor Thapsigargin, TRPM7 inhibitor NS-8593, T-type Ca2+ channels inhibitor Mibefradil, and Orai1 inhibitor GSK-7975A to understand the regulation of [Ca2+]i oscillations and mitochondrial activity during oocyte activation, and showed inbibition of SERCAs, TRPM7 and T-type Ca2+ channels caused Ca2+ signaling disturbances, mitochondrial activity and subsequent oocyte activation and ebryinic development.