Zoltan Machaty- Department of Animal Sciences Purdue University West Lafayette, West Lafayette, IN, United States
Embryo development is stimulated by calcium (Ca2+) signals that are generated in the egg cytoplasm by the fertilizing sperm. Eggs are formed via oogenesis. They go through a cell division known as meiosis, during which their diploid chromosome number is halved and new genetic combinations are created by crossing over. During formation the eggs also acquire cellular components that are necessary to produce the Ca2+ signal and also, to support development of the newly formed embryo. Ionized calcium is a universal second messenger used by cells in a plethora of biological processes and the eggs develop a “toolkit”, a set of molecules needed for signaling. Meiosis stops twice and these arrests are controlled by a complex interaction of regulatory proteins. The first meiotic arrest lasts until after puberty, when a luteinizing hormone surge stimulates meiotic resumption. The cell cycle proceeds to stop again in the middle of the second meiotic division, right before ovulation. The union of the female and male gametes takes place in the oviduct. Following gamete fusion, the sperm triggers the release of Ca2+ from the egg’s intracellular stores which in mammals is followed by repetitive Ca2+ spikes known as Ca2+ oscillations in the cytosol that last for several hours. Downstream sensor proteins help decoding the signal and stimulate other molecules whose actions are required for proper development including those that help to prevent the fusion of additional sperm cells to the egg and those that assist in the release from the second meiotic arrest, completion of meiosis and entering the first mitotic cell division. Here I review the major steps of egg formation, discuss the signaling toolkit that is essential to generate the Ca2+ signal and describe the steps of the signal transduction mechanism that activates the egg’s developmental program and turns it into an embryo.
1 Introduction
In sexual reproduction the union of the female and male gamete is a prerequisite for the generation of a new organism. The gametes must contain only half of the genetic material so that when they fuse together the diploid chromosome number is restored and the progeny will inherit genes from both parents. Also, during their formation the gametes must become capable of fertilization. This means that the spermatozoon must be able to reach the egg and activate its developmental program, while the egg must contain all the components of a signaling mechanism and all other molecules that are necessary to initiate embryo development. Development in the animal (and plant) kingdom is triggered by an increase in the cytosolic free calcium ion (Ca2+) concentration of the egg (Stricker, 1999). It is the sperm that stimulates the Ca2+ elevation and in mammalian species this Ca2+ signal takes the form of repetitive transients known as Ca2+ oscillations. In this review the formation of the mammalian egg will be described with special emphasis on the development of its signaling mechanism. The machinery that controls meiosis, the cell division of germ cells, will be explained briefly. The latest findings regarding the generation and maintenance of the Ca2+ signal will be presented and the major events that take place in response to the Ca2+ oscillations that culminate in the first mitosis of the newly formed embryo will be discussed. In this work the immature female gamete will be denoted as an oocyte. Once it completes meiotic maturation, extrudes the first polar body and arrests at the second meiotic metaphase it will be referred to as a mature oocyte or egg. For the purpose of this review the PubMed database was searched using keywords relevant to each section. Only those articles were included whose topic was related to the signaling process in the fertilized egg. I tried to provide a succinct story for each topic and make it readable and entertaining; if I left out anything or anybody, my apologies.
2 Oogenesis
2.1 Oocyte formation
Female gametes are highly specialized, terminally differentiated cells, whose single function is the propagation of life. They form in the fetal ovary through the process of oogenesis, from oogonia, which themselves develop from primordial germ cells. At one point during development oogonia enter meiosis, the cell division by which germ cells are produced. Meiosis encompasses one round of DNA replication followed by two rounds of chromosome segregation. The first round of meiosis is a reductional division, during which the homologous chromosomes (termed bivalents) are faithfully segregated so that only one copy of each chromosome remains in the oocyte and the other is discarded into the first polar body (Holt et al., 2013). Entry into meiosis involves a whole genome replication (S-phase) followed by a pairing of the homologous chromosomes. The homolog pairs then undergo double-strand DNA breakage during crossover, the “biological pinnacle of self-harm” (Sanders and Jones, 2018) and the subsequent repair of the breaks generates chromosomes made from both maternal and paternal components. In human germ cells about 2–3 crossovers take place between each pair of homologues (Alberts et al., 2002), resulting in a major shuffling of the genes. This increases genetic variation and thereby promotes evolutionary adaptation, which gives a huge advantage to sexual over asexual reproduction.
Meiosis is controlled primarily by the activity of a kinase, cyclin-dependent kinase 1 (Cdk1). In order to function as a kinase, Cdk1 requires the binding of cyclin B1, and together they form the maturation promoting factor (or M-phase promoting factor, MPF) with Cdk1 being the catalytic subunit and cyclin B1 the regulatory subunit. At this time MPF is inactive because Cdk1 is phosphorylated by an inhibitory enzyme, Wee1 and the cell is arrested at first prophase. Because of this meiotic arrest, the female germ cell, which is now a primary oocyte, spends the vast majority of its life at this state. Female mammals are born with a set of primary oocytes in their ovaries. The oocytes reside in primordial follicles, each ovary contains about 5,000 primordial follicles in mice (Bristol-Gould et al., 2006), 65,500 in sheep (Trounson et al., 1974), 66,500 in cattle (Erickson, 2010), 250,000 in pigs (Black and Erickson, 1968), and 500,000 in humans (Wallace and Kelsey, 2010). The germ cells at this stage have a large nucleus termed the germinal vesicle (GV), and they are arrested at the so-called GV stage, which is similar to the G2 phase of the mitotic cell cycle found in a somatic cell. The vast majority of these oocytes will undergo a process of cell death called atresia, the remainder will get periodically recruited into the growing follicle pool and will either be released from the follicle at ovulation or undergo atresia within non-ovulated atretic follicles (Holt et al., 2013).
2.2 Oocyte growth
At a certain stage during the reproductive cycle, oocyte growth is initiated. In the mouse, oocyte growth takes about 3 weeks and during this time the oocyte grows from 15 to 80 μm. In humans, growth takes approximately 6 months and the size of the oocyte increases from 35 to 120 μm. During growth, the oocyte accumulates all the transcripts essential for subsequent meiotic progression and fertilization (Sorensen and Wassarman, 1976). The cell cycle arrest is still maintained as the size of the oocyte, together with that of the follicle, increases. Limited Cdk1 and cyclin B1 availability seems to be a determining factor in the maintenance of this first meiotic arrest in many mammalian species (Fulka et al., 1988; Tatemoto and Horiuchi, 1995). However, as the oocyte grows, both proteins accumulate and acquire a greater capacity to associate with one another (de Vantéry et al., 1997). Once the oocyte reaches its final size and becomes meiotically competent, another regulatory system kicks in that helps to maintain the first meiotic arrest. This was demonstrated in rodents and cyclic adenosine 3′5′-monophosphate (cAMP) is the key player in the mechanism. cAMP is produced by the oocyte and its level is under the control of the surrounding granulosa cells (Hinckley et al., 2005). Granulosa cells produce another cyclic nucleotide, cGMP and after it is transported to the oocyte via gap junctions cGMP inhibits the hydrolysis (i.e., destruction) of cAMP (Zhang et al., 2010). cAMP keeps Cdk1 activity low by preventing its dephosphorylation (and thus activation), this blocks MPF activation and prevents entry into metaphase. The fully-grown oocyte also utilizes cyclin B1 degradation to keep Cdk1, and hence, MPF activity in check. Protein degradation is driven by the anaphase-promoting complex (APC), an E3 ubiquitin ligase that marks proteins for degradation by the 26S proteasome (Peters, 2006). APC requires the binding of a coactivator protein, either CDC20 or FZR1 (Holt et al., 2013). At this point it is FZR1 that controls the APC: oocytes lacking FZR1 undergo premature germinal vesicle breakdown (GVBD) in the presence of increasing cyclin B1 levels (Holt et al., 2011). FZR1-activated APC (APCFZR1) causes cyclin B1 degradation and in the absence of cyclin B1 Cdk1 cannot be activated, which leads to inactive MPF, and thus the first meiotic arrest is maintained until just before ovulation.
During growth, the primary oocyte acquires complex cytoplasmic organization and the cytoplasmic organelles become more abundant (Liu et al., 2006). In most species the centrioles disappear; the Balbiani body, a transient complex of diverse organelles forms around the nucleus; and the endoplasmic reticulum (ER) becomes more cortical. The number of ribosomes multiply, the Golgi apparatus enlarges and begins to export glycoproteins to form the zona pellucida and the cortical granules. The number of mitochondria, the organelle responsible for ATP production, also increases significantly. The mitochondrial DNA copy number, an indicator of the number of mitochondria in the cell, is 1,000 times higher in mature oocytes compared to primordial germ cells (Satouh and Sato, 2023). Morphology of the mitochondria also changes dramatically. At the time of oocyte formation, the diameter of the mitochondria in human oocytes is 1–1.5 μm with many cristae, whereas in fully grown oocytes the diameter is 0.4–0.6 μm with fewer cristae (Sathananthan and Trounson, 2000; Motta et al., 2000). All these events take place during the growth phase of oocyte development, which is extremely rapid and intense: mouse oocytes for example show a ∼300-fold increase in volume accompanied with a ∼300-fold rise in RNA content and a ∼38-fold elevation in the rate of protein synthesis (Wassarman and Albertini, 1994). Eventually, a gigantic germ cell is formed that is capable of becoming a viable embryo after fertilization.
2.3 Oocyte maturation
The final stage of oogenesis is oocyte maturation (or meiotic maturation). During maturation, the fully-grown oocyte acquires the ability to respond to the fertilizing sperm and produce the signal that initiates embryo development. The process involves two distinct yet interconnected events, nuclear and cytoplasmic maturation (Loutradis et al., 2006; Ferreira et al., 2009). Nuclear maturation encompasses germinal vesicle breakdown, chromosomal condensation and segregation, and extrusion of the first polar body, and as a result the diploid germ cell becomes haploid. Cytoplasmic maturation is less well defined but involves organelle reorganization, an increase in the content of intracellular Ca2+ stores and the storage of mRNAs and proteins that are required for the maturing oocyte and the early embryo prior to the activation of the embryonic genome (Yu et al., 2010).
The process is initiated by a luteinizing hormone (LH) surge from the pituitary gland that triggers the resumption of meiosis in the oocyte. The LH surge causes a decrease in NPR2 activity, the guanylyl cyclase that is responsible to produce cGMP in the cumulus cells. It also terminates gap junctions so the intercellular communication between the oocyte and the cumulus cells is lost. This results in reduced cGMP, and hence, low cAMP levels in the oocyte (Norris et al., 2008; Norris et al., 2009). Cdk1 is dephosphorylated (and thus activated) in the presence of low cAMP levels by the phosphatase called CDC25B (Lincoln et al., 2002); together this leads to high MPF levels that drive the cell cycle into metaphase I.
The increase in Cdk1 (and hence MPF) activity causes the dissolution of the nuclear lamins and subsequent germinal vesicle breakdown (GVBD; a G2/M transition), chromatin condensation and the formation of the first meiotic spindle (Sanders and Jones, 2018). In somatic cells, spindle formation is directed by the centrioles that promote the assembly of microtubules to form the bipolar spindle; oocytes lack centrioles and instead generate microtubule organizing centers (MTOCs) de novo that will guide spindle formation (Schuh and Ellenberg, 2007). The spindle apparatus harnesses each chromosome of the pair at random. The kinetochores of sister chromatids of each chromosome align side-by-side and orient towards the same pole, thus each homologue is separated from its partner and moved to opposite poles. This leads to a reductional cell division, i.e., the daughter cells end up with only half the number of chromosomes. The segregation of the chromosomes is controlled by the anaphase-promoting complex (APC), which is regulated, among other things, by the spindle assembly checkpoint (SAC). This mechanism prevents progress to anaphase until bivalents are properly captured on the spindle and has an important role in maintaining a high rate of faithful chromosome segregation in MI. The homologous chromosomes are held together by multisubunit protein rings called cohesins along the chromosome arms, these must be inactivated before chromosome segregation can proceed (Klein et al., 1999; Watanabe and Nurse, 1999). Inactivation is done by separase, an enzyme that is normally inhibited by securin. Once all the bivalents are correctly aligned and attached, SAC proteins allow for APC activation. Exit from metaphase also requires low MPF activity, which is achieved by cyclin B degradation, which is also controlled by APC. Rising Cdk1 activity promotes CDC20 to bind and activate the APC (Jin et al., 2010). Active APCCDC20 ubiquitinates cyclin B1 and thereby marks it for degradation (Hampl and Eppig, 1995); low MPF activity then facilitates anaphase entry. Activated APC also destroys securin, separase becomes active and hydrolyzes cohesin (importantly, centromeric cohesin holding the chromatids together is protected), and as a result homologous chromosomes can become separated (Herbert et al., 2003). During MI in oocytes, SAC activity is weak compared to its activity during mitosis, so chromosomal alignment abnormalities are often not detected (Nagaoka et al., 2011). This seems to be the reason for the high rates of incorrect bivalent segregation that leads to aneuploidy in oocytes. The cell cycle then progresses to telophase and upon completion of meiosis I a large secondary oocyte and a tiny first polar body are formed; the latter simply serves as the dumping ground for half of the chromosomes.
After the first round of meiosis a brief interphase known as interkinesis follows, during which no DNA replication takes place and no nuclear envelope forms, and the oocyte immediately begins the second round of meiosis. The cell cycle soon becomes arrested again, this time at the metaphase stage. The second meiotic arrest is sustained by high MPF activity, which blocks entry into anaphase. This is achieved by the inhibition of cyclin B1 destruction and an increase in Cdk1 activation. Cyclin B1 destruction is prevented because APC is inhibited by the early mitotic inhibitor 2 (Emi2; whose activity in turn is controlled by the cytostatic factor [CSF]), and also, because Cdk1 activity is kept at a high level via dephosphorylation by the phosphatase Cdc25A. In addition, APC inhibition also prevents the degradation of securin, so separase cannot hydrolyze cohesin and the bivalents cannot segregate (Sanders and Jones, 2018). This arrest happens shortly before ovulation, so that at ovulation mammalian eggs are released from the ovarian follicles while arrested at the MII stage. Maintaining the second meiotic arrest and preventing entry into the embryonic cell cycles without sperm makes “physiological sense” partly because term development of parthenogenetic embryos that form without the contribution of the male gamete would not be possible anyway due to the absence of crucial paternal genes, and also because this way the growth of potentially cancerous cells in the female genital tract can be prevented (Jones, 2007).
The intracellular organelles of the oocyte undergo significant reorganization during maturation. Some of the most prominent changes occur to the ER. In GV-stage mouse oocytes, the ER was found to be distributed throughout the cytoplasm with some of the ER accumulated around the germinal vesicle and as isolated ER clusters in the cytoplasm (Mehlmann et al., 1995; Kline et al., 1999; FitzHarris et al., 2003). During germinal vesicle breakdown, the ER becomes concentrated and forms a prominent ring around the germinal vesicle and then disperses throughout the cytoplasm. Finally, in mature oocytes the ER forms a fine reticular network throughout the cell with numerous dense accumulations (clusters) in the cortex. Although there are species-specific differences, this distribution of the ER in the cytoplasm serves a critical role during Ca2+ signaling at fertilization (Satouh and Sato, 2023). In addition, the sensitivity of the ER to Ca2+ release also increases during maturation. Concomitant with the ER reorganization during maturation, there is a 20%–90% increase in the immunoreactive mass of the inositol 1,4,5-trisphosphate receptors (IP3Rs) located in the ER membrane (Mehlmann et al., 1996; Fissore et al., 1999). In immature oocytes, the IP3Rs show homogeneous distribution in the entire cytoplasm except around the germinal vesicle. During maturation, the IP3Rs reorganize and become distributed in the cytoplasm in a more reticular manner with cortical clusters near the plasma membrane. This structural reorganization is thought to be important to acquire the ability to generate repetitive Ca2+ oscillations at fertilization (Mehlmann et al., 1996). The amount of Ca2+ stored in the ER also increases during maturation (Jones et al., 1995; Wakai et al., 2012). Due to the changes in these ER characteristics the Ca2+ signals that can be stimulated in the egg differ markedly depending on the maturation state of the female germ cell. Specifically, compared to Ca2+ signals found in eggs, those in immature oocytes have reduced amplitudes and durations, reduced sensitivity to IP3, lower oscillation frequencies, and the Ca2+ rises fail to propagate globally across the entire oocyte (reviewed by Stricker, 2006).
Mitochondria also undergo significant reorganization during maturation. These organelles provide energy in the form of ATP, which is critically important for the maturing oocyte (Stojkovic et al., 2001). During oocyte growth and the early phases of maturation, the oocyte is connected to cumulus cells that provide pyruvate to be used by the germ cell’s mitochondria to generate ATP, they can also supply the oocyte with ready-made ATP. Following ovulation however, the egg becomes uncoupled from the supporting somatic cells and must rely on its own mitochondria to generate ATP (Downs, 1995; Johnson et al., 2007). Mitochondrial activity also shows unique changes during oocyte maturation. ATP content increases significantly in pig (Brevini et al., 2005), bovine (Stojkovic et al., 2001) and mouse (Yu et al., 2010) oocytes during maturation and it is correlated with developmental success (Stojkovic et al., 2001; Van Blerkom et al., 1995). In immature oocytes, the mitochondria have a more peripheral position and at germinal vesicle breakdown they aggregate around the germinal vesicle as a ring-like structure. After the germinal vesicle breaks down the mitochondria disperse throughout the cytoplasm (Dumollard et al., 2006; Yu et al., 2010). This redistribution of mitochondria is accompanied with three distinct increases in cytosolic ATP levels in mouse oocytes that are associated with discrete events of oocyte maturation, with the third peak corresponding to polar body extrusion (Yu et al., 2010). The number of mitochondria also increases during oocyte development. Primordial germ cells soon after formation contain less than 10 mitochondria and by the time they become oogonia this number increases to 200. The number of mitochondria in primary oocytes is about 6,000, which then jumps up to more than 100,000 by the end of cytoplasmic maturation (Trimarchi et al., 2000; Cummins, 2004).
The localization dynamics of the ER follow a similar pattern as that of the mitochondria, which suggests a functional link between the two organelles. A prominent example of this inter-organelle coordination is observed in mouse oocytes. GV-stage oocytes are reported to undergo spontaneous Ca2+ oscillations, during which Ca2+ is periodically released from the ER into the cytosol (Wakai and Fissore, 2019). The released Ca2+ is then absorbed and stored by the mitochondria and this is believed to stimulate mitochondrial ATP production. The Golgi apparatus, a delivery center and the site for protein processing, also undergoes redistribution. The Golgi receives proteins and lipids from the ER and exports them to endosomes, lysosomes, or the plasma membrane. In GV-stage oocyte the Golgi apparatus is dispersed throughout the cytoplasm and at GVBD it undergoes drastic fragmentation (Moreno et al., 2002). Cortical granules are formed from the Golgi complex. In the GV-stage oocyte the granules are distributed in clusters throughout the cytoplasm and by the end of maturation they localize in the cortex, below the plasma membrane (Sathananthan et al., 1985). Cortical granules are secretory granules that have important roles during fertilization to prevent polyspermy (Liu, 2011). Overall, the changes during maturation, a process that begins with germinal vesicle breakdown and ends when the cell cycle is arrested in metaphase of the second meiotic division, empowers the egg to interact appropriately with the fertilizing sperm.
3 The signaling toolkit
Early during the course of evolution, cells of living organisms selected ionized calcium for signaling (Clapham, 2007). Signaling was essential to adapt to an ever-changing environment and for the purpose they needed messengers. Requirements for such messengers were that 1) their concentration must be able to fluctuate with time, i.e., their level in resting cells must be low but they need to be rapidly produced and released when needed, and 2) they must be capable of influencing the function of proteins. Ca2+ can fulfill both requirements. Cells spend a lot of energy to keep their intracellular Ca2+ levels approximately 20,000 times lower than the extracellular environment and thus they can generate a signal by rapidly increasing the Ca2+ level in the cytosol. Also, protein function depends on shape and charge, and Ca2+ can change these features by binding to the proteins. Because of these characteristics, Ca2+ became the most ubiquitous signal transduction element in living organisms involved in a wide range of biological processes. In neurons, skeletal muscle and heart muscle neurotransmitters trigger a Ca2+ influx to induce changes; antigen stimulation of immune cells leads to a Ca2+ influx that promotes an immune response against the invading pathogens; and Ca2+ entry through sperm-specific CatSper channels in the sperm tail induces hyperactivated motility prior to fertilization, just to name a few (reviewed by Cai et al., 2015).
Although it is essential for signaling, extended exposure to high Ca2+ levels is detrimental to the cell, probably because Ca2+ precipitates phosphate, another essential component of cellular physiology. Because of this, cells chelate, compartmentalize, or extrude Ca2+ (Clapham, 2007). Calcium chelators can protect cells from a toxic Ca2+ overload; a common chelator of Ca2+ in cells is the EF hand domain, which is present in a large number of proteins (Nakayama and Kretsinger, 1994). In eukaryotic cells the smooth ER is the main Ca2+ storage compartment, often occupying more than 10% of the cell volume (Lam and Galione, 2013). Ca2+ accumulates in its lumen where it can reach a concentration of more than 100 µM. In the ER, Ca2+ is stored attached to luminal Ca2+-binding proteins, these can be classified as either buffers or chaperones. Buffer proteins including calsequestrin and calreticulin simply bind Ca2+ with high capacity and hold it in the ER; chaperones such as calnexin assist in protein folding and quality control while they might also have a role in modulating Ca2+ signaling (Berridge, 2002). The ER actively sequesters Ca2+ into its lumen using SERCA (smooth endoplasmic reticulum Ca2+ ATP-ase) pumps that use ATP for the work. The SERCA pump family is encoded by three different genes (SERCA 1, SERCA 2, and SERCA 3), and from these genes seven different isoforms are expressed (SERCA 1a/1b, SERCA 2a/2b, and SERCA 3a/3b/3c) (Periasamy and Kalyanasundaram, 2007). In mouse eggs the presence of SERCA2b protein was demonstrated (Wakai et al., 2013). Finally, an increasing amount of experimental data indicate that other organelles such as mitochondria and the Golgi apparatus are also able to sequester Ca2+ and thus play a role in Ca2+ homeostasis and signaling (Bagur and Hajnóczky, 2017). The mitochondrial calcium uniporter (MCU) and secretory-pathway calcium ATPase (SPCA) can mediate Ca2+ uptake into these organelles (Duchen, 2000; Wu et al., 2023). Ca2+ uptake by the mitochondria not only assists in Ca2+ buffering but also promotes ATP synthesis, and this Ca2+-driven ATP production seems to be a major contribution of mitochondria to the Ca2+ homeostasis in mammalian eggs (Hajnóczky et al., 1995). By measuring intracellular and mitochondrial Ca2+ levels in mouse eggs simultaneously it has been found that the Ca2+ spikes are accompanied by parallel increases in the concentration of mitochondrial Ca2+, which stimulates ATP production (Dumollard et al., 2008). It has subsequently been shown that this Ca2+-driven ATP output is important to power the SERCA pumps during refilling of the ER (Wakai et al., 2013).
During signaling, the Ca2+ stored in the ER is released via two types of Ca2+ release channel receptors, IP3Rs and ryanodine receptors. In mammalian cells the IP3R predominates (reviewed by Berridge, 2009). The receptors have four subunits that span the ER membrane and form the channel pore. There are three IP3R isoforms (IP3R1, IP3R2, and IP3R3) with slightly different properties; mammalian eggs overwhelmingly express IP3R1, although they have the other isoforms as well (Parrington et al., 1998; Fissore et al., 1999). The opening of the IP3R requires binding by IP3 and Ca2+, low cytosolic Ca2+ levels stimulate the discharge of the stored Ca2+, whereas high levels inhibit it (Iino, 1990).
Once a Ca2+ rise is generated, the resting intracytoplasmic Ca2+ level must be restored. Some of the cytosolic Ca2+ is pumped back into the stores by the SERCA pumps described above, while the rest is removed from the cell through the plasma membrane. This is done primarily by the plasma membrane Ca2+ ATP-ase (PMCA) pumps that use ATP to pump Ca2+ against the immense concentration gradient to move Ca2+ out of the cell (reviewed by Carafoli, 1991). In addition, Na+/Ca2+ exchangers (NCX) located in the plasma membrane can move one Ca2+ out of the cell in exchange for three Na+ (Blaustein and Lederer, 1999). There is evidence for the function of both mechanisms in eggs (Carroll, 2000; Machaty et al., 2002b). In somatic cells another membrane protein, the Na+/Ca2+-K+ exchanger (NCKX) has also been described, this exchanger cotransports one Ca2+ with one K+ in exchange for four Na+ (Altimimi and Schnetkamp, 2007).
The removal of Ca2+ from the cell can be so effective that an influx mechanism may be needed to restore intracellular Ca2+ levels for proper cell function. Cells can use a number of mechanisms to accomplish this including receptor-operated channels (ROCs), voltage-gated channels (VGCs), transient receptor potential (TRP) channels, and ORAI channels (reviewed by Wakai et al., 2019). Two classes of voltage-gated calcium channels have been described. High-voltage-gated channels such as P/Q, N, R and L type channels are activated by strong depolarization. Low-voltage-gated channels in the other class require lower depolarization for opening, these are known as T-type channels (or CaV3.1-CaV3.3) (Arikkath and Campbell, 2003). The presence of CaV3.2 was demonstrated in mouse eggs (Peres, 1986; Bernhardt et al., 2015). It was found that GV-stage oocytes show larger CaV3.2-mediated currents compared to mature eggs. In addition, mice lacking CaV3.2 channels were still able to display the Ca2+ oscillations at fertilization indicating that the function of these channels is to mediate the Ca2+ influx in GV-stage oocytes and prepare them to become capable of mounting the repetitive Ca2+ transients at fertilization. Because mouse eggs show only a slight change in the membrane potential during fertilization (Igusa et al., 1983; Jaffe and Cross, 1984) it remains a conundrum why these voltage-gated channels are expressed and functional in these cells. The presence of TRP channels have also been shown in mammalian eggs (Machaty et al., 2002a; Carvacho et al., 2013). In somatic cells such channels mediate Ca2+ entry in response to a great number of stimuli during the perception of pain, temperature, different kinds of taste, pressure, and vision. They are known to be modulated by protein kinase C (PKC) (Mandadi et al., 2011). In mouse eggs the expression of TRPV3 and TRPM7 was demonstrated and it was shown that the expression level changes during the course of maturation (Carvacho et al., 2013; Carvacho et al., 2016). Finally, components of the store-operated Ca2+ entry (SOCE) mechanism are also present in mammalian oocytes and eggs. In many non-excitable cells Ca2+ influx across the plasma membrane is stimulated in response to store depletion (Putney, 1990). One major SOCE component is the STIM protein, which is located in the ER membrane and serves as a sensor of Ca2+ content (Liou et al., 2005). Upon store depletion, STIM proteins oligomerize, translocate to plasma membrane-adjacent regions and open the other major SOCE component, the ORAI channel, which leads to Ca2+ influx (Feske et al., 2006). Two STIM proteins (STIM1 and STIM2) and three ORAI proteins (ORAI1, ORAI2, and ORAI3) have been identified in mammalian cells. The presence of STIM1 and ORAI1 has also been demonstrated in porcine and mouse eggs (Koh et al., 2009; Yu et al., 2009; Wang et al., 2012).
Changes in Ca2+ concentration inside of cells can be detected by Ca2+ sensor (also known as trigger or adaptor) proteins. These Ca2+ binding proteins have the ability to bind Ca2+ and undergo a significant conformational change that allows them to bind specific target proteins and modify their function. The best studied of these proteins is the ubiquitous Ca2+ sensor calmodulin. This protein changed little during the course of 1.5 billion years of evolution, clearly demonstrating the evolutionary importance of Ca2+ signaling (Clapham, 1995; Clapham, 2007). Equipped with four EF-hand Ca2+-binding motifs calmodulin amplifies the small size of Ca2+ to the scale of proteins by changing its own conformation upon Ca2+ binding. In fertilized eggs, calmodulin mediates the effects of Ca2+ by activating Ca2+/calmodulin-dependent protein kinase II (CaMKII). Another calmodulin target, the protein phosphatase calcineurin has also been described in T cells of the immune system. Calcineurin serves a key role in activating the transcription factor called nuclear factor of activated T cells (NFAT) by dephosphorylating it (Rusnak and Mertz, 2000). The presence and arrangement of the different components of the signaling toolkit in the egg allows for the creation and maintenance of the Ca2+ signal that the fertilizing sperm generates to stimulate embryo development.
4 The sperm protein that activates the egg
The fundamental question regarding fertilization is: how does the sperm activate the egg? That egg activation is a chemical process and it involves changes in the concentration of ions in the egg cytoplasm was first proposed by Jacques Loeb towards the end of the 19th century (Loeb, 1899). He based his idea on the observation that sea urchin eggs underwent parthenogenetic activation after being held in seawater that contained increased levels of ions. It was later established that the fertilizing sperm activates the egg’s developmental program by stimulating an increase in its cytosolic free Ca2+ concentration (Mazia, 1937; Ridgway et al., 1977). The major question then became one of how the sperm causes the Ca2+ increase in the egg. It became clear that the Ca2+ came from internal sources and it was also established that IP3 stimulated its release (Whitaker and Irvine, 1984; Busa et al., 1985; Miyazaki, 1988). Following decades of intensive research, the prevailing hypothesis was that the sperm binds to a transmembrane receptor on the surface of the egg and by doing so it stimulates the enzyme phospholipase C to generate IP3 (Shilling et al., 1994). However, the egg surface receptor to which the sperm can bind, and whose stimulation would lead to IP3 production and Ca2+ release have never been found. The other major line of thinking was the so-called fusion hypothesis. Experiments with mouse and sea urchin eggs revealed that following binding of the sperm to the egg plasma membrane, fusion of the two gametes took place prior to Ca2+ release (McCulloh and Chambers, 1992; Lawrence et al., 1997). The obvious explanation to this was that the sperm might contain a factor that diffuses into the egg cytoplasm and causes Ca2+ release.
Interestingly, it was also Loeb who first suggested that the sperm contains some factor that causes egg activation (Loeb, 1913). He claimed that the sperm delivers two substances to the egg, one of which (a “lysin”) acts on the surface to form a fertilization envelope, and another factor whose role is to prevent degeneration of the egg following formation of the fertilization envelope. Although many of his statements today are hard to reconcile with our current understanding of egg activation, Loeb was fascinated by the way embryo development is stimulated and made outstanding contributions to the field. The first experimental evidence that the sperm may contain such a factor came from experiments that demonstrated that the microinjection of a sperm extract into sea urchin eggs led to the formation of the fertilization envelope, an indication of egg activation (Dale et al., 1985). Later it was demonstrated that extracts from mammalian sperm could also induce activation of mouse, rabbit and hamster eggs (Swann, 1990; Stice and Robl, 1990). Importantly, the mammalian sperm extracts were also able to trigger repetitive Ca2+ oscillations in eggs of various species including hamster, mouse, human, bovine and pig (Swann, 1990; Swann, 1994; Homa and Swann, 1994; Wu et al., 1997; Machaty et al., 2000). The generation of repetitive oscillations is important because several stimuli can cause an increase in the cytosolic Ca2+ concentration but triggering a series of Ca2+ spikes was only possible by using spermatozoa. Another discovery that supported the sperm factor hypothesis was the successful application of intracytoplasmic sperm injection (ICSI) to treat male factor infertility (Palermo et al., 1992). As it was demonstrated later, injecting the sperm into the egg cytoplasm caused repetitive Ca2+ oscillations that could not be mimicked by sham injections (Tesarik et al., 1994; Nakano et al., 1997), further supporting the theory that the sperm activates the egg via a diffusible factor and not by binding to a cell surface receptor. The egg activating factor was shown to be located in the sperm head, in the perinuclear matrix (Kimura et al., 1998).
An important step in the identification of the sperm factor came when it was discovered that mammalian sperm extracts contained the enzyme phospholipase C that was active enough to generate IP3 to cause Ca2+ oscillations (Jones et al., 1998). This indicated that the sperm factor could be a PLC, although as follow-up studies determined it was unlike any of the known PLC isoforms. The factor was eventually identified as a novel PLC isoform termed PLCζ (PLCzeta), a sperm-specific protein (Saunders et al., 2002). It is present in the sperm of many different mammalian species and its ability to cause Ca2+ oscillations is evolutionary conserved (Cox et al., 2002; Rogers et al., 2004; Kurokawa et al., 2005; Yoon and Fissore, 2007). Its gene is present in all mammalian genomes sequenced to-date, and also in some birds and fish (Swann, 2022). PLCζ has been shown to induce Ca2+ oscillations in mouse, human, pig, and cow eggs (reviewed by Swann, 2023). The protein is localized in the sperm head and diffuses into the egg after sperm-egg fusion, this explains why microinjection of the sperm into the egg during intracytoplasmic sperm injection causes Ca2+ oscillations (Sato et al., 1999). It is also present in cytosolic sperm extracts that were shown to stimulate repetitive Ca2+ transients after injection into eggs (Saunders et al., 2002). The specific location of this protein is within the post-acrosomal region of the sperm, which is where gamete fusion begins. PLCζ is present in this region at a concentration that can stimulate repetitive Ca2+ transients in the egg (the amount of PLCζ in a mouse spermatozoon is 20–50 fg and the microinjection of 4–8 fg of the protein has been shown to be capable of inducing Ca2+ oscillations) (Saunders et al., 2002).
The molecular structure of PLCζ has been determined in great detail. PLCζ has a molecular weight of ∼70 kDa and is the smallest of all PLC isoforms (Thanassoulas et al., 2022). The enzyme lacks the N-terminal pleckstrin homology (PH) domain that is present in other PLC isoforms, and instead contains four N-terminal EF hand domains, X and Y catalytic domains separated by the XY-linker, and a C2 domain at the C-terminus. The EF hands contain Ca2+ binding residues and provide the enzyme with high Ca2+ sensitivity; deletion or mutation of these conserved Ca2+ binding residues abolish the Ca2+-induced activity of PLCζ. In effect, the EF hands act as molecular switches controlled by Ca2+ spikes. The X and Y catalytic domains are responsible for the catalysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2); a mutation in this region causes a loss in the enzyme’s ability to generate IP3 and trigger Ca2+ oscillations (Nomikos et al., 2011). C2 domains are known as Ca2+-dependent membrane-targeting modules involved in targeting proteins to phospholipid-containing cell membranes (Nalefski and Falke, 1996; Zheng et al., 2000). Interestingly, the C2 domain of PLCζ has no predicted Ca2+ binding site, nevertheless deletion of this domain abolishes the ability of PLCζ to induce Ca2+ oscillations (Nomikos et al., 2005). It was proposed that it facilitates the anchoring of the enzyme to membranes through its interaction with specific phosphoinositides (Thanassoulas et al., 2022). Finally, the XY-linker plays multiple roles in influencing PLCζ action. As mentioned above, unlike other isoforms PLCζ does not have a PH domain that usually functions to bind PIP2 within biological membranes. It is believed however, that positively charged residues within the XY-linker target the enzyme to PIP2, possibly via electrostatic interactions (Nomikos et al., 2007). This idea is supported by the finding that a decrease in the net positive charge of the XY-linker resulted in a decline in the enzyme’s ability to bind PIP2 or to stimulate Ca2+ transients after microinjection into eggs (Nomikos et al., 2011). The XY-linker also contains a predicted nuclear localization signal (NLS) sequence, which is a potential binding site for the nuclear transport receptor (NTR); this sequence may be important in the regulation of PLCζ function, at least in the mouse (Larman et al., 2004). A great number of mutations in the PLCζ gene have been described in patients with egg activation deficiencies. The mutations affected various domains of the protein (but primarily the X and Y catalytic domains) leading to fertilization failure, this highlights the importance of PLCζ in fertilization (reviewed by Thanassoulas et al., 2022).
Eggs of different species have different sensitivities to PLCζ. For example, mouse eggs are much more sensitive to human PLCζ than human eggs, the difference in sensitivity is about 30-fold (Yu et al., 2008). In fact, mouse eggs are about 10 times more sensitive to IP3-induced Ca2+ release as well compared to human eggs (Storey et al., 2021). This implies that something in mouse eggs sensitizes IP3Rs that is absent in human eggs. It has recently been proposed that the factor that regulates IP3Rs may be the concentration of ATP (Swann, 2023). Somatic cells express mostly IP3R2s and IP3R3s while mammalian eggs possess primarily IP3R1s. IP3R1s differ significantly in the way they are regulated by ATP: their open probability increases in response to ATP, whereas IP3R3s need about 10 times higher ATP levels to open and IP3R2s are not gated by ATP (Tu et al., 2005). This suggests that mammalian eggs are highly sensitive to ATP modulation. The IP3R1 was shown to be sensitive to the Mg2+-free form, or ATP4−. Approximately 95% of the ATP in the cytoplasm is bound to Mg2+ as MgATP2−, which is the form that supplies the energy to many biological processes (Mak et al., 1999). However, the total ATP concentration in mouse eggs was found to be ∼3.3 mM (Storey et al., 2021), so the ATP4− level in such eggs seems to be high enough (∼200 µM) to modulate the opening of IP3R1s. In contrast, the total ATP concentration in human eggs is about 1.4 mM (which is equivalent to ∼90 µM of ATP4−), and hamster eggs may have even lower ATP levels (Van Blerkom et al., 2003). Thus, ATP levels seem to be different in the eggs of various mammalian species and this may explain their different sensitivity to PLCζ.
Other sperm proteins have also been proposed as possible candidate factors, these include the postacrosomal sheath WW domain-binding protein (PAWP; Wu et al., 2007), a truncated c-kit tyrosine kinase (Sette et al., 2002), and an extramitochondrial form of citrate synthase (Harada et al., 2007). These proteins have been suggested to trigger Ca2+ release and subsequent activation in mouse and human eggs. However, independent laboratories have been unable to verify the reported results and until then it is difficult to accept these candidate proteins as sperm proteins with a role in fertilization (Swann, 2023). Finally, there seems to exist a secondary factor in the sperm that may serve as a backup mechanism. This factor is observable only when PLCζ is absent but its exact identity remains elusive. When mouse eggs were fertilized in vitro with sperm lacking PLCζ, they showed 1 to 4 Ca2+ transients that occurred approximately 1 h after the sperm fused to their plasma membrane (Hachem et al., 2017; Nozawa et al., 2018). It is puzzling how these sperm can generate such Ca2+ spikes and the possibility exists that they contain a second factor which, when introduced into the egg, generates IP3 and causes the release of stored Ca2+. Interestingly, in the same studies, when PLCζ KO sperm were injected into the eggs via intracytoplasmic sperm injection, no Ca2+ oscillations occurred. Thus, the second factor seems to work during IVF but not during ICSI. In addition, the putative second factor’s effect is markedly slower: when the eggs were fertilized with sperm lacking PLCζ, the Ca2+ oscillations were initiated with a distinct delay of approximately 1 h following sperm-egg fusion. The belated Ca2+ transients caused delayed cortical granule exocytosis and as a result many zygotes were polyspermic. Identification of this putative second sperm factor will hopefully help in understanding these rather strange characteristics. Although questions remain that need to be answered, accumulating evidence shows that PLCζ is currently the only moiety that successfully fulfills all the requirements to be considered the predominant mammalian egg-activating factor.
5 Calcium signaling at fertilization
In most species, a rise in the intracellular free Ca2+ concentration is triggered by the fertilizing sperm and forms the stimulus for the egg-embryo transition. Without fertilization the eggs typically die within 48 h, but the fertilizing sperm triggers a series of physiological and biochemical changes that causes activation of the oocyte’s developmental program. The Ca2+ signal is evolutionarily conserved, which is striking considering the fact that eggs of different species arrest at different stages during meiosis (Stricker, 1999). Ca2+ is both necessary and sufficient for egg activation at fertilization in mammals (Kline and Kline, 1992a). PLCζ delivered into the egg by the sperm hydrolyzes PIP2 into IP3 and diacylglycerol (DAG). IP3 then binds to its receptor on the surface of the ER, which leads to the release of stored Ca2+ into the cytosol, while DAG can activate the enzyme PKC. In lower species there is a single Ca2+ rise that crosses the egg starting at the site of sperm fusion; this single Ca2+ elevation initiates embryo development. In mammalian species however, the initial Ca2+ rise is followed by a series of transients that last for several hours, this was first described by Cuthbertson and Cobbold (1985). The long-lasting stimulus seems to be needed to properly activate the egg. There are two major events that are triggered by Ca2+: cortical granule exocytosis to prevent the fusion of additional sperm cells to the egg, and the stimulation of cell cycle resumption. Results of experiments where the number of Ca2+ spikes generated in the eggs was tightly controlled revealed that in order to trigger complete cortical granule exocytosis and to induce exit from the MII arrest, multiple transients were needed (Lawrence et al., 1998; Ducibella et al., 2002; Ozil et al., 2005). In addition, fewer transients were needed to stimulate cortical granule release than cell cycle resumption, probably because it is physiologically more urgent to prevent the entry of additional sperm cells than it is to stimulate the completion of meiosis.
In mouse and hamster eggs it has been shown that the initial Ca2+ rise takes the form of a wave of Ca2+ release that crosses the egg; the wave starts at the point where the sperm fused to the egg and crosses the egg in about 5–10 s (Miyazaki et al., 1986; Deguchi et al., 2000). Earlier it was believed that after the first 2 to 3 transients the Ca2+ rises begin synchronously in the entire cytoplasm. However, it was later determined that subsequent Ca2+ elevations are also waves, but the waves are increasingly faster (after about 15 min the waves cross the egg in ∼1 s) and their point of origin in the cortex varies from one transient to the other (Deguchi et al., 2000). The reason for this shift in the wave initiation point is probably the diffusion of PLCζ in the egg cytoplasm. During the first Ca2+ wave, PLCζ is localized in the area where the sperm fused with the egg plasma membrane and this is the point from which the wave originates. The molecular weight of PLCζ is ∼70 kDa and proteins of this size require about 10 min to cross the mouse egg by diffusion (Swann, 1996). After ∼10 min PLCζ will have diffused throughout the entire cytoplasm, which explains why the wave initiation point varies in the cortex. These subsequent waves also have a higher speed, which is attributable to the unique way PIP2 is distributed in the egg cytoplasm. As mentioned above, PLCζ generates the Ca2+-releasing second messenger IP3 by hydrolyzing PIP2. PIP2 is typically localized in the plasma membrane of cells but interestingly, in mouse eggs, most PIP2 is found to be present in small vesicles that are distributed throughout the entire cytoplasm (Yu et al., 2012; Sanders et al., 2018). It is not entirely clear why PIP2 is present in these cytoplasmic vesicles but it may be related to the mechanism by which Ca2+ oscillations are maintained in eggs. The initial Ca2+ spikes at fertilization can be described by so-called IP3-based models; such models involve the positive feedback of Ca2+ on the IP3R (Sneyd et al., 2006; Politi et al., 2006). Mouse and human eggs have mostly IP3R1s (Miyazaki et al., 1992; Lee et al., 2010), which show a bell-shaped sensitivity to Ca2+ when stimulated by IP3 (Foskett et al., 2007). The IP3R opens in response to IP3 and the opening is enhanced by increasing levels of Ca2+. This Ca2+-enhanced Ca2+ release operates for the initial part of the Ca2+ transient but the IP3R closes when the Ca2+ concentration in the cytosol reaches the micromolar range. However, it has been demonstrated in mouse eggs that later sperm-induced Ca2+ oscillations can be explained by a different model, the Ca2+-induced regenerative IP3 formation model (Sanders et al., 2018). According to this model, PLCζ generates IP3 that causes Ca2+ release from the ER. In turn Ca2+ stimulates further PLCζ activity (PLCζ contains four EF hand domains that are responsible for the protein’s high sensitivity to Ca2+; (Nomikos et al., 2005)), this leads to more IP3 production and hence more Ca2+ release. So, the Ca2+ oscillations at fertilization are maintained by PLCζ through a positive feedback mechanism based upon Ca2+-induced IP3 generation (Swann, 2023). The model also explains the importance of PIP2 located in cytoplasmic vesicles. If PIP2 hydrolysis happened exclusively at the plasma membrane, the relatively slow diffusion of IP3 would limit the speed of the propagating Ca2+ wave, which, as mentioned above, reaches the speed of about 1 sec a few minutes after sperm-egg fusion. The ER is spread throughout the egg cytoplasm and is in close proximity with the PIP2 vesicles that are themselves about 2 µm apart from one another (Sanders et al., 2018). With this arrangement the IP3 generated is within a few micrometers from the IP3Rs, this short distance facilitates the rapid release of Ca2+ from the stores and the rapid propagation of the Ca2+ wave. The finding that in fertilized mouse eggs there is no detectable PIP2 hydrolysis in the plasma membrane also supports the notion that PLCζ generates IP3 from cytoplasmic vesicle-resident PIP2 (Yu et al., 2012).
The level of IP3 also oscillates in phase with Ca2+ following gamete fusion. When changes in the IP3 concentration was monitored in mouse eggs using the IP3 indicator IRIS-2.3TMR, it was found that there was a small monotonic IP3 increase during the first few Ca2+ spikes, followed by distinct oscillations in IP3 after approximately 20 min into the series of Ca2+ transients (Matsu-Ura et al., 2019). This shift is consistent with the diffusion characteristics of PLCζ. During the first few transients, PLCζ is localized in the area where the sperm has fused to the egg plasma membrane and the Ca2+ spikes will primarily be dependent on IP3 diffusion and IP3 stimulation. After about 20 min, PLCζ diffuses throughout the entire cytoplasm and the Ca2+ transients will be dependent on Ca2+-induced IP3 production, with IP3 levels oscillating in synchrony with Ca2+ oscillations (Swann, 2023).
As it was discussed above, Ca2+-stimulated PLCζ hydrolyzes PIP2 to generate IP3 and IP3 binds its receptor on the ER to induce the release of stored Ca2+. At one point during Ca2+ release the increase in the cytosolic Ca2+ level is terminated but interestingly, it is not entirely clear how. The nature of the negative feedback that decreases the intracytoplasmic Ca2+ concentration is somewhat vague (Swann, 2023). There are several mathematical models to describe Ca2+ oscillations in somatic cells; some models propose that it is the complete depletion of the ER stores that terminates Ca2+ release. However, in mouse eggs it was shown that complete emptying of the stores does not happen during each Ca2+ transient (Wakai et al., 2013; Sanders et al., 2018). Other models propose that the Ca2+ release is terminated by the Ca2+-induced desensitization of the IP3R (Sneyd et al., 2006; Politi et al., 2006). The fact that at high IP3 levels - which is expected when PLCζ is present - Ca2+ is not effective in closing the IP3R (Mak et al., 1988) argues against this theory. Thus, the exact mechanism responsible for terminating the Ca2+ transients remains elusive.
The sperm-induced Ca2+ oscillations last for several hours, in mice they stop around the time of pronucleus formation when the cell cycle enters interphase (Marangos et al., 2003; Larman et al., 2004). Evidence has shown that in fertilized mouse eggs PLCζ is sequestered into the pronuclei and this is responsible for the cessation of Ca2+ transients. In fact, mouse pronuclei have oocyte activating ability due to PLCζ sequestration. This was demonstrated when nuclei from 1- and 2-cell-stage fertilized mouse embryos were transferred to unfertilized eggs, the transferred (pro)nuclei caused repetitive Ca2+ transients, the completion of meiosis and pronuclear formation. Nuclei from parthenogenetic embryos did not trigger egg activation indicating that the ability to cause activation was specific to nuclei transferred from fertilized embryos. In addition, there are a short series of Ca2+ transients during the first mitosis in the mouse embryo; these are stimulated shortly after nuclear envelope breakdown and believed to be the result of PLCζ being released from the pronuclei (Kono et al., 1995). Sequence analysis of PLCζ from all species studied has shown that the protein possesses a putative nuclear localization signal in the XY-linker region (Thanassoulas et al., 2022); this sequence facilitates the accumulation of mouse PLCζ in the newly formed pronuclei. Although it is tempting to suggest that PLCζ sequestration regulates the cessation of the Ca2+ signal in mammalian eggs, this does not seem to be the case. The sperm-induced Ca2+ oscillations did not stop when the pronuclei formed in fertilized bovine eggs and continued during pronucleus migration and nuclear envelope breakdown (Nakada et al., 1995). Furthermore, following cRNA microinjection into the egg cytoplasm, human, rat, medaka fish and horse PLCζ did not translocate to the pronucleus in mouse zygotes (Ito et al., 2008; Sato et al., 2013), and bovine PLCζ failed to accumulate in the pronucleus of either mouse or bovine zygotes (Cooney et al., 2010). Thus, PLCζ sequestration into the pronuclei may serve a role in the termination of the Ca2+ oscillations in mouse zygotes but a different mechanism may be at play in other mammals.
Another phenomenon also seems to be important in the cessation of the Ca2+ oscillations. It was reported that in mouse eggs IP3R1s are downregulated and more than 50% of their mass is lost by the time pronuclei form (Brind et al., 2000; Jellerette et al., 2000). The degradation is due to the persistent stimulation of the phosphoinositide pathway and the continuous exposure of the receptors to IP3. Ultimately, the degradation of the IP3Rs, which appears to be mediated by the ubiquitin-proteasome pathway, is responsible for the diminished periodicity of the Ca2+-oscillations as the egg approaches interphase (Lee et al., 2010). By that time however, it has fulfilled its role and stimulated all the events that are associated with egg activation.
6 Ca2+ influx
An influx of Ca2+ across the egg plasma membrane is also required to maintain the Ca2+ transients. It is well documented that in Ca2+-free media the Ca2+ oscillations, either sperm-induced or triggered by intracytoplasmic injection of PLCζ, slow down or stop entirely (Igusa and Miyazaki, 1983; Wakai et al., 2013). An indication that an influx of Ca2+ is generated because of the release of Ca2+ from the ER came from experiments using thapsigargin, a SERCA pump inhibitor. Adding thapsigargin to mouse eggs blocks the SERCA pumps. Ca2+ slowly leaks out of the ER but the pumps are not able move Ca2+ back so the stores become depleted; this stimulates a Ca2+ entry across the plasma membrane (Kline and Kline, 1992b). It has been proposed that the Ca2+ influx was responsible for reloading the intracellular stores (Miyazaki, 1991). A similar phenomenon has been described in somatic cells as well. Agonist stimulation in numerous non-excitable cells stimulates Ca2+ release from the ER followed by an influx of Ca2+ across the plasma membrane (Putney, 1990; Berridge, 1990). The Ca2+ influx pathway is activated by store depletion and is termed store-operated Ca2+ entry (SOCE, previously known as capacitative Ca2+ entry). The Ca2+ influx regulated by the intracellular Ca2+ stores has been demonstrated in fertilized mouse eggs (McGuinness et al., 1996), while artificially induced SOCE has been shown to be present in pig and human eggs (Machaty et al., 2002a; Martín-Romero et al., 2008).
Although the mechanism of the Ca2+ influx in eggs is well-established and its importance during fertilization has been clearly demonstrated, the components of the pathway has remained elusive. The breakthrough in the understanding of the SOCE pathway in somatic cells came when the stromal interaction molecule 1 (STIM1) protein was identified as the sensor of ER Ca2+ content that links store depletion to Ca2+ influx (Liou et al., 2005; Roos et al., 2005). The channel component of the SOCE pathway, ORAI1, was identified soon afterwards (Feske et al., 2006; Vig et al., 2006). The presence and role of these proteins in mediating SOCE during fertilization has been demonstrated in pig eggs. When STIM1 levels are downregulated in porcine eggs prior to fertilization using siRNA, the eggs show only a single Ca2+ transient; additional Ca2+ rises cannot be detected in eggs that lack STIM1 (Lee et al., 2012). Similarly, downregulation of ORAI1 expression by siRNA microinjection also appears to block the sperm-induced Ca2+ oscillations (Wang et al., 2012). The Ca2+ entry mediated by STIM1 and ORAI1 has been found to be essential to sustain the fertilization Ca2+ signal in pig eggs (Wang et al., 2015), and Förster Resonance Energy Transfer (FRET) analysis has clearly indicated the repetitive interaction between STIM1 and ORAI1 during fertilization, in synchrony with the sperm-induced Ca2+ transients (Zhang et al., 2018). These data strongly imply that store-operated Ca2+ entry, mediated by an interplay between STIM1 and ORAI1 operate during fertilization in the pig.
Intriguingly, in mouse eggs the Ca2+ entry that maintains the sperm-induced Ca2+ oscillations is mediated by a different mechanism. In this species, inhibiting SOCE by specific inhibitors (Miao et al., 2012), or by the expression of protein fragments that interfere with the interaction between STIM1 and ORAI1 (Takahashi et al., 2013) does not prevent the Ca2+ transients at fertilization. Furthermore, in transgenic mice that lack STIM1, STIM2, or ORAI1, eggs of the knockout animals display normal Ca2+ influx after store depletion and importantly, show normal patterns of Ca2+ oscillations at fertilization (Bernhardt et al., 2017). Combined, these data indicate that as opposed to pigs, in mouse eggs SOCE is not needed during fertilization. Research then focused on TRP channels as these can serve as Ca2+ influx channels in several cell types (Ramsey et al., 2006), they are expressed in eggs (Petersen et al., 1995; Machaty et al., 2002a), and because certain TRP isoforms are regulated by PKC, a protein that is activated during fertilization (Hardie, 2007). An ion channel current activated by TRP agonists has been identified in mouse eggs; the current is absent in eggs of transgenic animals lacking transient receptor potential vanilloid member 3 (TRPV3) channels. However, eggs of TRPV3-knockout animals display normal Ca2+ oscillations upon fertilization (Carvacho et al., 2013) revealing that these channels are not essential for normal fertilization. The T-type channel CaV3.2 has also been suggested to be a part of the Ca2+ entry mechanism. Mouse eggs lacking the α1 subunit of this channel show impaired Ca2+ oscillations at fertilization (Bernhardt et al., 2015) indicating their involvement in Ca2+ homeostasis and signaling. However, mice having non-functional CaV3.2 channels are still fertile (although they show impaired fertility), which implies that additional influx mechanisms must have a role in maintaining the Ca2+ oscillations at fertilization. More promising results have been obtained when TRPV3 was knocked out together with CaV3.2; deletion of these channels has disrupted Ca2+ store refilling and has a negative effect on the sperm-induced Ca2+ transients (Mehregan et al., 2021). The Transient Receptor Potential Melastanin 7 (TRPM7)-like channel has also been identified as a potential candidate. Such channels are present in mouse eggs and their inhibition prevents spontaneous Ca2+ influxes in immature oocytes, while it also disrupts the repetitive sperm-induced Ca2+ signal (Carvacho et al., 2016; Bernhardt et al., 2017). Finally, mouse eggs lacking both TRPM7 and CaV3.2 are unable to maintain the long-lasting Ca2+ oscillations at fertilization and it has been concluded that together they almost completely account for the majority of Ca2+ influx following store depletion (Bernhardt et al., 2018). In addition, TRPM7 may also have a role in mediating Mg2+ homeostasis as it has been demonstrated that fertilization is more effective (Herrick et al., 2015) and the sperm-induced Ca2+ signal is more efficiently generated in the presence of low Mg2+ levels (Zhang and Machaty, 2017; Ozil et al., 2017).
Although the underlying mechanisms are different, a Ca2+ influx is essential in both mouse and pig eggs to maintain the Ca2+ oscillations at fertilization. The generally accepted reasoning is that the Ca2+ influx is needed to refill the intracellular stores, which in turn is important for the next Ca2+ transient to take place. Lately however, in light of the Ca2+ sensitivity of PLCζ another explanation has been offered. It has been suggested that the rapid cessation of the Ca2+ transients in the absence of extracellular Ca2+ may not be due to low Ca2+ levels in the ER but due to reduced levels of Ca2+ in the cytosol (Swann, 2023). Too low cytosolic Ca2+ levels may be insufficient to stimulate PLCζ to generate IP3. This postulation is supported by the results of an experiment using thapsigargin, the SERCA pump inhibitor. As mentioned above, incubating eggs in the presence of thapsigargin leads to the slow leak of stored Ca2+ from the ER into the cytoplasm. In the experiment, PLCζ was injected into mouse eggs to generate a series of Ca2+ transients in their cytoplasm. When the eggs were subsequently placed in Ca2+-free medium the oscillations stopped as expected. However, when thapsigargin was added to the holding medium the oscillations resumed and continued for more than an hour (Sanders et al., 2018). This indicates that mouse eggs can undergo repetitive Ca2+ transients in Ca2+-free medium if the cytosolic Ca2+ concentration is raised; the Ca2+ in the cytosol can stimulate PLCζ to generate IP3 and additional Ca2+ spikes are generated. This alternative explanation regarding the effect of the Ca2+ influx also highlights its importance in maintaining the Ca2+ oscillations during fertilization.
7 Cortical granule exocytosis
Ca2+ stimulates a major event of egg activation, cortical granule exocytosis, the release of the content of the cortical granules into the perivitelline space to prevent polyspermy. The process seems to be induced by a signal transduction cascade that most probably involves myosin light chain kinase (MLCK). MLCKs are Ca2+/calmodulin-dependent protein kinases. Following its release from the ER, Ca2+ binds the intracellular Ca2+-binding protein calmodulin, and the complex can activate a number of downstream protein kinases including MLCK. Once activated, MLCK phosphorylates (i.e., activates) the light chain of myosin and activated myosin will be able to interact with actin (Simerly et al., 1998; Kamm and Stull, 2001). MLCK and myosin are involved in the regulation of cytoskeletal-mediated events in the egg cortex indicated by the fact that ML-7, an MLCK inhibitor is able to block chromatin-induced cortical actin localization and lateral cortical granule movements in mouse eggs (Deng et al., 2005). It was also found that blocking MLCK with ML-7, or using blebbistatin, a myosin II isoform-specific inhibitor, did not decrease the activity of CaMKII and had no effect on the elevation of intracellular Ca2+ levels, but markedly reduced cortical granule exocytosis (Matson et al., 2006).
It has been suggested that exocytosis of the cortical granule contents alters the properties of the zona pellucida, thus prevents the entry of excess sperm into the perivitelline space. The enzyme responsible for transforming the zona pellucida is believed to be astacin-like metalloendopeptidase (Astl, also known as ovastacin; Burkart et al., 2012). Astl partially degrades the ZP2 protein, a component of the zona pellucida and thereby blocks the passage of additional sperm cells. During mouse in vitro fertilization, cortical reaction and ZP2 cleavage (zona hardening) reached a plateau about 40 min after the sperm-egg fusion, once the first 2 or 3 Ca2+ transients are completed (Satouh et al., 2017). In eggs of Astl-deficient mice the sperm cells accumulated in the perivitelline space indicating a lack of zona hardening (although curiously, no polyspermy was observed in in vivo fertilized eggs). During the course of this so-called zona reaction another zona pellucida glycoprotein, ZP3 is also modified and it has been found that as a result it loses its sperm receptor activity and the ability to induce the acrosome reaction (Wassarman, 1988). These changes in the structure of the zona pellucida prevent additional sperm from reaching the egg plasma membrane and ultimately guarantee normal (i.e., diploid) chromosome number in the embryo.
Interestingly, the release of the content of granules of a different type was also reported to play a role at fertilization. These granules are also located beneath the plasma membrane and have high Zn2+ content. As it was revealed through a series of experiments the Zn2+ stored in the granules is released into the extracellular milieu during the Ca2+ oscillations (Kim et al., 2011). Artificial depletion of the Zn2+ stores by treating the eggs with a Zn2+ chelator caused activation of mouse and porcine eggs in the absence of Ca2+ spikes indicating the importance of intracellular Zn2+ in the second meiotic arrest of the eggs (Suzuki et al., 2010; Uh et al., 2019). These so-called zinc fluxes seem to regulate meiosis through the APC inhibitor Emi2, a zinc-binding protein; zinc chelators strip Zn2+ from Emi2, thus causing meiotic resumption (Bernhardt et al., 2012).
8 Completion of meiosis
Another major event triggered by Ca2+ is the resumption and eventual completion of meiosis. This process is mediated via a signal transduction mechanism that is different from the one that stimulates cortical granule exocytosis. As mentioned above, Ca2+ released from the ER binds calmodulin, which in this case activates the Ca2+ sensing enzyme called calmodulin-dependent protein kinase II (CaMKII). The activity of CAMKII is oscillatory and parallels the transient elevations of Ca2+ in fertilized eggs (Markoulaki et al., 2003; Markoulaki et al., 2004). CaMKII in turn activates Wee1b, a protein tyrosine kinase, which inhibits Cdk1 by phosphorylation (Oh et al., 2011). CamKII also phosphorylates and thus inhibits the APC inhibitor Emi2 through Polo-like kinase 1 (Plk1; Solc et al., 2015) and as Emi2 is degraded, APC is activated (Madgwick et al., 2006). This enables the destruction of cyclin B1 as well as securin, thus stimulating cell cycle progression (Madgwick and Jones, 2007). The inhibition of Cdk1 and destruction of cyclin B1 means MPF inactivation. Interestingly, according to a recent study, fertilization causes MPF inactivation through the inhibition of Cdk1 activity without concomitant cyclin B1 degradation in bovine eggs, so species-specific differences may exist (Valencia et al., 2021). At the same time the decrease in securin results in the activation of separase, which in turn hydrolyzes centromeric cohesin that leads to the separation of sister chromatids at anaphase (Fulka et al., 1994). As it involves the segregation of sister chromatids, this second cell division is an equational division, similar to mitosis (Sanders and Jones, 2018). It is also asymmetric (just like the first round of meiosis was), leading to the formation of a one-cell embryo and a second polar body. Full completion of meiosis II requires the reformation of the nuclear envelope and chromosome decondensation, which means that another signaling component, MAPK also needs to be destroyed. MAPK levels are high during the MII arrest and high levels of MAPK activity were shown to be incompatible with the presence of a pronuclear envelope (Moos et al., 1996). The repetitive Ca2+ oscillations cause the destruction of MAPK and thus meiosis II is allowed to be completed and the pronuclei are formed (Gonzalez-Garcia et al., 2014).
A single rise of cytosolic Ca2+ is believed to be a poor activator of mammalian eggs, probably because in such eggs Emi2 is only transiently degraded. These eggs may complete their second meiotic division and extrude a second polar body but tend to re-arrest at a new metaphase without forming a pronucleus (Kubiak, 1989). When the activity of Emi2 is regained it will inhibit the APC, and as a result cyclin B1 is reaccumulated that leads to active MPF and metaphase arrest (Jones, 2007). It was suggested that in aged eggs, those that have passed the window of their normal time of fertilization, the situation is different (Fulton and Whittingham, 1978). Such eggs might be less able to maintain high MPF levels and in their case a single monotonic Ca2+ rise can cause egg activation effectively. Therefore, under physiological conditions the importance of the oscillatory Ca2+ signal may be to provide a signal that is long enough to guarantee that the egg exits meiosis (Jones, 2005). Another function of the repetitive signal seems to be its effect on long-term embryo development. When the eggs were exposed to a series of Ca2+ rises of varying amplitude and duration in a specifically designed activation chamber, they all underwent high rates of activation (Ducibella et al., 2006). However, their longer-term (i.e., post-implantation) development was markedly affected by the Ca2+ spiking regiment, with more transients having a positive effect on chromatin remodeling and reprogramming of gene expression. It is hypothesized that the repetitive Ca2+ spikes are important not only to stimulate the degradation of proteins whose function is to maintain the metaphase II arrest, but they may also be critical to switch on the expression of proteins whose function is needed for proper embryo development (Jones, 2007).
9 Conclusion
A lot has been discovered about the making of an embryo in the previous decades. The egg forms during oogenesis and by the time it reaches the site of fertilization, it acquires all the components and features that allow it to appropriately respond to the fertilizing sperm. It must be activated to become an embryo and it is clear that activation is mediated by a signaling mechanism that involves Ca2+. This important second messenger is released from the intracellular stores of the egg and once in the cytoplasm it interacts with downstream components of the signaling cascade that results in the exclusion of additional sperm cells to prevent polyspermy, the completion of the meiotic division and the initiation of the first mitotic cell cycle of the newly formed embryo. PLCζ emerged as the moiety that the sperm uses to generate the Ca2+ signal. Although it is of critical importance in the process of mammalian fertilization, there are still a number of things that need to be learned about PLCζ. For example, the putative binding partners of PLCζ within the sperm remain to be elucidated; identification of those molecules is a prerequisite to completely understand the function of this important egg-activating molecule. The most promising candidate for this role seems to be calmodulin (Leclerc et al., 2020). Calmodulin is the primary sensor of intracellular Ca2+ levels and it is able to interact with numerous proteins including several phospholipase isoforms. It is localized in similar regions of the sperm as PLCζ and the XY-linker region of PLCζ contains an amino acid sequence that is recognized by calmodulin (Thanassoulas et al., 2022). Future studies will determine the importance of such a PLCζ-calmodulin complex in the regulation of PLCζ function. In addition, the precise regulation of PLCζ activity in the egg cytoplasm is also unknown. PLCζ stimulates repetitive Ca2+ transients only in eggs and not in somatic cells (Phillips et al., 2011) and it is unclear how this is achieved. Mammalian PLCs are typically activated by interaction with other proteins, either in the cell where they are produced or in those to which they are transported (Suh et al., 2008). It is possible that a unique “egg factor” exists that interacts with PLCζ in the egg cytoplasm and enables it to induce multiple Ca2+ oscillations but the identity of such a factor has not been defined yet. Another distinctive feature of PLCζ is that it targets PIP2 in intracellular vesicles and not in the plasma membrane (Yu et al., 2012). This seems to be important to facilitate the propagation of the Ca2+ wave throughout the cytoplasm but the way it is regulated is currently unclear. It is possible that PLCζ identifies these vesicles by interacting with the yet-to-be identified egg factor, which may be a cytosolic binding protein or a membrane receptor (Swann and Lai, 2013). Finally, the role of ATP in the signaling process at fertilization is not completely understood although it may profoundly affect the ability of the egg to generate the signal that turns the egg into an embryo.
Author contributions
ZM: Writing–original draft, Writing–review and editing.
Funding
The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.
Conflict of interest
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). Molecular biology of the cell. 4th Edition. New York: Garland Science.
Altimimi, H. F., and Schnetkamp, P. P. (2007). Na+/Ca2+-K+ exchangers (NCKX): functional properties and physiological roles. Channels (Austin). 1, 62–69. doi:10.4161/chan.4366
Arikkath, J., and Campbell, K. P. (2003). Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr. Opin. Neurobiol. 13, 298–307. doi:10.1016/s0959-4388(03)00066-7
Bagur, R., and Hajnóczky, G. Y. (2017). Intracellular Ca2+ sensing: its role in calcium homeostasis and signaling. Mol. Cell. 66, 780–788. doi:10.1016/j.molcel.2017.05.028
Bernhardt, M. L., Kong, B. Y., Kim, A. M., O'Halloran, T. V., and Woodruff, T. K. (2012). A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes. Biol. Reprod. 86, 114. doi:10.1095/biolreprod.111.097253
Bernhardt, M. L., Padilla-Banks, E., Stein, P., Zhang, Y., and Williams, C. J. (2017). Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs. Cell Calcium 65, 63–72. doi:10.1016/j.ceca.2017.02.004
Bernhardt, M. L., Stein, P., Carvacho, I., Krapp, C., Ardestani, G., Mehregan, A., et al. (2018). TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization. Proc. Natl. Acad. Sci. U. S. A. 115, E10370–E10378. doi:10.1073/pnas.1810422115
Bernhardt, M. L., Zhang, Y., Erxleben, C. F., Padilla-Banks, E., McDonough, C. E., Miao, Y. L., et al. (2015). CaV3.2 T-type channels mediate Ca2⁺ entry during oocyte maturation and following fertilization. J. Cell. Sci. 128, 4442–44452. doi:10.1242/jcs.180026
Berridge, M. J. (1990). Calcium oscillations. J. Biol. Chem. 265, 9583–9586. doi:10.1016/s0021-9258(19)38704-6
Berridge, M. J. (2002). The endoplasmic reticulum: a multifunctional signaling organelle. Cell Calcium 32, 235–249. doi:10.1016/s0143416002001823
Berridge, M. J. (2009). Inositol trisphosphate and calcium signalling mechanisms. Biochim. Biophys. Acta. 1793, 933–940. doi:10.1016/j.bbamcr.2008.10.005
Black, J. L., and Erickson, B. H. (1968). Oogenesis and ovarian development in the prenatal pig. Anat. Rec. 161, 45–55. doi:10.1002/ar.1091610105
Blaustein, M. P., and Lederer, W. J. (1999). Sodium/calcium exchange: its physiological implications. Physiol. Rev. 79, 763–854. doi:10.1152/physrev.1999.79.3.763
Brevini, T. A., Vassena, R., Francisci, C., and Gandolfi, F. (2005). Role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. Biol. Reprod. 72, 1218–1223. doi:10.1095/biolreprod.104.038141
Brind, S., Swann, K., and Carroll, J. (2000). Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca2+ or egg activation. Dev. Biol. 223, 251–265. doi:10.1006/dbio.2000.9728
Bristol-Gould, S. K., Kreeger, P. K., Selkirk, C. G., Kilen, S. M., Mayo, K. E., Shea, L. D., et al. (2006). Fate of the initial follicle pool: empirical and mathematical evidence supporting its sufficiency for adult fertility. Dev. Biol. 298, 149–154. doi:10.1016/j.ydbio.2006.06.023
Burkart, A. D., Xiong, B., Baibakov, B., Jiménez-Movilla, M., and Dean, J. (2012). Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. J. Cell. Biol. 197, 37–44. doi:10.1083/jcb.201112094
Busa, W. B., Ferguson, J. E., Joseph, S. K., Williamson, J. R., and Nuccitelli, R. (1985). Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores. J. Cell. Biol. 101, 677–682. doi:10.1083/jcb.101.2.677
Cai, X., Wang, X., Patel, S., and Clapham, D. E. (2015). Insights into the early evolution of animal calcium signaling machinery: a unicellular point of view. Cell Calcium 57, 166–173. doi:10.1016/j.ceca.2014.11.007
Carafoli, E. (1991). Calcium pump of the plasma membrane. Physiol. Rev. 71, 129–153. doi:10.1152/physrev.1991.71.1.129
Carroll, J. (2000). Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation. J. Reprod. Fertil. 118, 337–342. doi:10.1530/jrf.0.1180337
Carvacho, I., Ardestani, G., Lee, H. C., McGarvey, K., Fissore, R. A., and Lykke-Hartmann, K. (2016). TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse. Sci. Rep. 6, 34236. doi:10.1038/srep34236
Carvacho, I., Lee, H. C., Fissore, R. A., and Clapham, D. E. (2013). TRPV3 channels mediate strontium-induced mouse-egg activation. Cell. Rep. 5, 1375–1386. doi:10.1016/j.celrep.2013.11.007
Cooney, M. A., Malcuit, C., Cheon, B., Holland, M. K., Fissore, R. A., and D'Cruz, N. T. (2010). Species-specific differences in the activity and nuclear localization of murine and bovine phospholipase C zeta 1. Biol. Reprod. 83, 92–101. doi:10.1095/biolreprod.109.079814
Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002). Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes. Reproduction 124, 611–623. doi:10.1530/rep.0.1240611
Cummins, J. M. (2004). The role of mitochondria in the establishment of oocyte functional competence. Eur. J. Obstet. Gynecol. Reprod. Biol. 115 (Suppl. 1), S23–S29. doi:10.1016/j.ejogrb.2004.01.011
Cuthbertson, K. S., and Cobbold, P. H. (1985). Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+. Nature 316, 541–542. doi:10.1038/316541a0
Dale, B., DeFelice, L. J., and Ehrenstein, G. (1985). Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction. Experientia 41, 1068–1070. doi:10.1007/BF01952148
Deguchi, R., Shirakawa, H., Oda, S., Mohri, T., and Miyazaki, S. (2000). Spatiotemporal analysis of Ca2+ waves in relation to the sperm entry site and animal-vegetal axis during Ca2+ oscillations in fertilized mouse eggs. Dev. Biol. 218, 299–313. doi:10.1006/dbio.1999.9573
Deng, M., Williams, C., and Schultz, R. M. (2005). Role of MAP kinase and myosin light-chain kinase in chromosome-induced development of mouse egg polarity. Dev. Biol. 278, 358–366. doi:10.1016/j.ydbio.2004.11.013
de Vantéry, C., Stutz, A., Vassalli, J. D., and Schorderet-Slatkine, S. (1997). Acquisition of meiotic competence in growing mouse oocytes is controlled at both translational and posttranslational levels. Dev. Biol. 187, 43–54. doi:10.1006/dbio.1997.8599
Downs, S. M. (1995). The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev. Biol. 167, 502–512. doi:10.1006/dbio.1995.1044
Duchen, M. R. (2000). Mitochondria and calcium: from cell signalling to cell death. J. Physiol. 529 (Pt 1), 57–68. doi:10.1111/j.1469-7793.2000.00057.x
Ducibella, T., Huneau, D., Angelichio, E., Xu, Z., Schultz, R. M., Kopf, G. S., et al. (2002). Egg-to- embryo transition is driven by differential responses to Ca2+ oscillation number. Dev. Biol. 250, 280–291. doi:10.1006/dbio.2002.0788
Ducibella, T., Schultz, R. M., and Ozil, J. P. (2006). Role of calcium signals in early development. Semin. Cell. Dev. Biol. 17, 324–332. doi:10.1016/j.semcdb.2006.02.010
Dumollard, R., Campbell, K., Halet, G., Carroll, J., and Swann, K. (2008). Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes. Dev. Biol. 316, 431–440. doi:10.1016/j.ydbio.2008.02.004
Dumollard, R., Duchen, M., and Sardet, C. (2006). Calcium signals and mitochondria at fertilisation. Semin. Cell. Dev. Biol. 17, 314–323. doi:10.1016/j.semcdb.2006.02.009
Erickson, B. H. (2010). Development and senescence of the postnatal bovine ovary. J. Anim. Sci. 25, 800–805. doi:10.2527/jas1966.253800x
Ferreira, E. M., Vireque, A. A., Adona, P. R., Meirelles, F. V., Ferriani, R. A., and Navarro, P. A. (2009). Cytoplasmic maturation of bovine oocytes: structural and biochemical modifications and acquisition of developmental competence. Theriogenology 71, 836–848. doi:10.1016/j.theriogenology.2008.10.023
Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S. H., Tanasa, B., et al. (2006). A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179–185. doi:10.1038/nature04702
Fissore, R. A., Longo, F. J., Anderson, E., Parys, J. B., and Ducibella, T. (1999). Differential distribution of inositol trisphosphate receptor isoforms in mouse oocytes. Biol. Reprod. 60, 49–57. doi:10.1095/biolreprod60.1.49
FitzHarris, G., Marangos, P., and Carroll, J. (2003). Cell cycle-dependent regulation of structure of endoplasmic reticulum and inositol 1,4,5-trisphosphate-induced Ca2+ release in mouse oocytes and embryos. Mol. Biol. Cell. 14, 288–301. doi:10.1091/mbc.e02-07-0431
Foskett, J. K., White, C., Cheung, K. H., and Mak, D. O. (2007). Inositol trisphosphate receptor Ca2+ release channels. Physiol. Rev. 87, 593–658. doi:10.1152/physrev.00035.2006
Fulka, J., Fléchon, J. E., Motlík, J., and Fulka, J. (1988). Does autocatalytic amplification of maturation-promoting factor (MPF) exist in mammalian oocytes? Gamete Res. 21, 185–192. doi:10.1002/mrd.1120210209
Fulka, J., Moor, R. M., and Fulka, J. (1994). Sister chromatid separation and the metaphase-anaphase transition in mouse oocytes. Dev. Biol. 165, 410–417. doi:10.1006/dbio.1994.1263
Fulton, B. P., and Whittingham, D. G. (1978). Activation of mammalian oocytes by intracellular injection of calcium. Nature 273, 149–151. doi:10.1038/273149a0
Gonzalez-Garcia, J. R., Bradley, J., Nomikos, M., Paul, L., Machaty, Z., Lai, F. A., et al. (2014). The dynamics of MAPK inactivation at fertilization in mouse eggs. J. Cell Sci. 127 (Pt 12), 2749–2760. doi:10.1242/jcs.145045
Hachem, A., Godwin, J., Ruas, M., Lee, H. C., Ferrer Buitrago, M., Ardestani, G., et al. (2017). PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence. Development 144, 2914–2924. doi:10.1242/dev.150227
Hajnóczky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995). Decoding of cytosolic calcium oscillations in the mitochondria. Cell 82, 415–424. doi:10.1016/0092-8674(95)90430-1
Hampl, A., and Eppig, J. J. (1995). Analysis of the mechanism(s) of metaphase I arrest in maturing mouse oocytes. Development 121, 925–933. doi:10.1242/dev.121.4.925
Harada, Y., Matsumoto, T., Hirahara, S., Nakashima, A., Ueno, S., Oda, S., et al. (2007). Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster. Dev. Biol. 306, 797–808. doi:10.1016/j.ydbio.2007.04.019
Hardie, R. C. (2007). TRP channels and lipids: from Drosophila to mammalian physiology. J. Physiol. 578, 9–24. doi:10.1113/jphysiol.2006.118372
Herbert, M., Levasseur, M., Homer, H., Yallop, K., Murdoch, A., and McDougall, A. (2003). Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1. Nat. Cell. Biol. 5, 1023–1025. doi:10.1038/ncb1062
Herrick, J. R., Strauss, K. J., Schneiderman, A., Rawlins, M., Stevens, J., Schoolcraft, W. B., et al. (2015). The beneficial effects of reduced magnesium during the oocyte-to-embryo transition are conserved in mice, domestic cats and humans. Reprod. Fertil. Dev. 27, 323–331. doi:10.1071/RD13268
Hinckley, M., Vaccari, S., Horner, K., Chen, R., and Conti, M. (2005). The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes. Dev. Biol. 287, 249–261. doi:10.1016/j.ydbio.2005.08.019
Holt, J. E., Lane, S. I., and Jones, K. T. (2013). The control of meiotic maturation in mammalian oocytes. Curr. Top. Dev. Biol. 102, 207–226. doi:10.1016/B978-0-12-416024-8.00007-6
Holt, J. E., Tran, S. M., Stewart, J. L., Minahan, K., García-Higuera, I., Moreno, S., et al. (2011). The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes. Development 138, 905–913. doi:10.1242/dev.059022
Homa, S. T., and Swann, K. (1994). A cytosolic sperm factor triggers calcium oscillations and membrane hyperpolarizations in human oocytes. Hum. Reprod. 9, 2356–2361. doi:10.1093/oxfordjournals.humrep.a138452
Igusa, Y., and Miyazaki, S. (1983). Effects of altered extracellular and intracellular calcium concentration on hyperpolarizing responses of the hamster egg. J. Physiol. 340, 611–632. doi:10.1113/jphysiol.1983.sp014783
Igusa, Y., Miyazaki, S., and Yamashita, N. (1983). Periodic hyperpolarizing responses in hamster and mouse eggs fertilized with mouse sperm. J. Physiol. 340, 633–647. doi:10.1113/jphysiol.1983.sp014784
Iino, M. (1990). Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the Guinea pig taenia caeci. J. Gen. Physiol. 95, 1103–1122. doi:10.1085/jgp.95.6.1103
Ito, M., Shikano, T., Oda, S., Horiguchi, T., Tanimoto, S., Awaji, T., et al. (2008). Difference in Ca2+ oscillation-inducing activity and nuclear translocation ability of PLCZ1, an egg-activating sperm factor candidate, between mouse, rat, human, and medaka fish. Biol. Reprod. 78, 1081–1090. doi:10.1095/biolreprod.108.067801
Jaffe, L. A., and Cross, N. L. (1984). Electrical properties of vertebrate oocyte membranes. Biol. Reprod. 30, 50–54. doi:10.1095/biolreprod30.1.50
Jellerette, T., He, C. L., Wu, H., Parys, J. B., and Fissore, R. A. (2000). Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation. Dev. Biol. 223, 238–250. doi:10.1006/dbio.2000.9675
Jin, F., Hamada, M., Malureanu, L., Jeganathan, K. B., Zhou, W., Morbeck, D. E., et al. (2010). Cdc20 is critical for meiosis I and fertility of female mice. PLoS Genet. 6, e1001147. doi:10.1371/journal.pgen.1001147
Johnson, M. T., Freeman, E. A., Gardner, D. K., and Hunt, P. A. (2007). Oxidative metabolism of pyruvate is required for meiotic maturation of murine oocytes in vivo. Biol. Reprod. 77, 2–8. doi:10.1095/biolreprod.106.059899
Jones, K. T. (2005). Mammalian egg activation: from Ca2+ spiking to cell cycle progression. Reproduction 130, 813–823. doi:10.1530/rep.1.00710
Jones, K. T. (2007). Intracellular calcium in the fertilization and development of mammalian eggs. Clin. Exp. Pharmacol. Physiol. 34, 1084–1089. doi:10.1111/j.1440-1681.2007.04726.x
Jones, K. T., Carroll, J., and Whittingham, D. G. (1995). Ionomycin, thapsigargin, ryanodine, and sperm induced Ca2+ release increase during meiotic maturation of mouse oocytes. J. Biol. Chem. 270, 6671–6677. doi:10.1074/jbc.270.12.6671
Jones, K. T., Cruttwell, C., Parrington, J., and Swann, K. (1998). A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca2+ release in sea urchin egg homogenates. FEBS Lett. 437, 297–300. doi:10.1016/s0014-5793(98)01254-x
Kamm, K. E., and Stull, J. T. (2001). Dedicated myosin light chain kinases with diverse cellular functions. J. Biol. Chem. 276, 4527–4530. doi:10.1074/jbc.R000028200
Kim, A. M., Bernhardt, M. L., Kong, B. Y., Ahn, R. W., Vogt, S., Woodruff, T. K., et al. (2011). Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs. ACS Chem. Biol. 6, 716–723. doi:10.1021/cb200084y
Kimura, Y., Yanagimachi, R., Kuretake, S., Bortkiewicz, H., Perry, A. C., and Yanagimachi, H. (1998). Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol. Reprod. 58, 1407–1415. doi:10.1095/biolreprod58.6.1407
Klein, F., Mahr, P., Galova, M., Buonomo, S. B. C., Michaelis, C., Nairz, K., et al. (1999). A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98, 91–103. doi:10.1016/S0092-8674(00)80609-1
Kline, D., and Kline, J. T. (1992a). Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev. Biol. 149, 80–89. doi:10.1016/0012-1606(92)90265-i
Kline, D., and Kline, J. T. (1992b). Thapsigargin activates a calcium influx pathway in the unfertilized mouse egg and suppresses repetitive calcium transients in the fertilized egg. J. Biol. Chem. 267, 17624–17630. doi:10.1016/S0021-9258(19)37088-7
Kline, D., Mehlmann, L., Fox, C., and Terasaki, M. (1999). The cortical endoplasmic reticulum (ER) of the mouse egg: localization of ER clusters in relation to the generation of repetitive calcium waves. Dev. Biol. 215, 431–442. doi:10.1006/dbio.1999.9445
Koh, S., Lee, K., Wang, C., Cabot, R. A., and Machaty, Z. (2009). STIM1 regulates store-operated Ca2+ entry in oocytes. Dev. Biol. 330, 368–376. doi:10.1016/j.ydbio.2009.04.007
Kono, T., Carroll, J., Swann, K., and Whittingham, D. G. (1995). Nuclei from fertilized mouse embryos have calcium-releasing activity. Development 121, 1123–1128. doi:10.1242/dev.121.4.1123
Kubiak, J. Z. (1989). Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest. Dev. Biol. 136, 537–545. doi:10.1016/0012-1606(89)90279-0
Kurokawa, M., Sato, K., Wu, H., He, C., Malcuit, C., Black, S. J., et al. (2005). Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm. Dev. Biol. 285, 376–392. doi:10.1016/j.ydbio.2005.06.029
Lam, A. K., and Galione, A. (2013). The endoplasmic reticulum and junctional membrane communication during calcium signaling. Biochim. Biophys. Acta. 1833, 2542–2559. doi:10.1016/j.bbamcr.2013.06.004
Larman, M. G., Saunders, C. M., Carroll, J., Lai, F. A., and Swann, K. (2004). Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCζ. J. Cell Sci. 117, 2513–2521. doi:10.1242/jcs.01109
Lawrence, Y., Ozil, J. P., and Swann, K. (1998). The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases. Biochem. J. 335 (Pt 2), 335–342. doi:10.1042/bj3350335
Lawrence, Y., Whitaker, M., and Swann, K. (1997). Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse. Development 124, 233–241. doi:10.1242/dev.124.1.233
Leclerc, P., Goupil, S., Rioux, J. F., Lavoie-Ouellet, C., Clark, M. È., Ruiz, J., et al. (2020). Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa. J. Cell. Physiol. 235, 5340–5352. doi:10.1002/jcp.29421
Lee, B., Yoon, S. Y., Malcuit, C., Parys, J. B., and Fissore, R. A. (2010). Inositol 1,4,5-trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations. J. Cell. Physiol. 222, 238–247. doi:10.1002/jcp.21945
Lee, K., Wang, C., and Machaty, Z. (2012). STIM1 is required for Ca2+ signaling during mammalian fertilization. Dev. Biol. 367, 154–162. doi:10.1016/j.ydbio.2012.04.028
Lincoln, A. J., Wickramasinghe, D., Stein, P., Schultz, R. M., Palko, M. E., De Miguel, M. P., et al. (2002). Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation. Nat. Genet. 30, 446–449. doi:10.1038/ng856
Liou, J., Kim, M. L., Heo, W. D., Jones, J. T., Myers, J. W., Ferrell, J. E., et al. (2005). STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241. doi:10.1016/j.cub.2005.05.055
Liu, K., Rajareddy, S., Liu, L., Jagarlamudi, K., Boman, K., Selstam, G., et al. (2006). Control of mammalian oocyte growth and early follicular development by the oocyte PI3 kinase pathway: new roles for an old timer. Dev. Biol. 299, 1–11. doi:10.1016/j.ydbio.2006.07.038
Liu, M. (2011). The biology and dynamics of mammalian cortical granules. Reprod. Biol. Endocrinol. 9, 149. doi:10.1186/1477-7827-9-149
Loeb, J. (1899). On the nature of the process of fertilization and the artificial production of normal larvæ (plutei) from the unfertilized eggs of the sea urchin. Am. J. Physiol. 3, 135–138. doi:10.1152/ajplegacy.1899.3.3.135
Loutradis, D., Kiapekou, E., Zapanti, E., and Antsaklis, A. (2006). Oocyte maturation in assisted reproductive techniques. Ann. N. Y. Acad. Sci. 1092, 235–246. doi:10.1196/annals.1365.020
Machaty, Z., Bonk, A. J., Kühholzer, B., and Prather, R. S. (2000). Porcine oocyte activation induced by a cytosolic sperm factor. Mol. Reprod. Dev. 57, 290–295. doi:10.1002/1098-2795(200011)57:3<290::AID-MRD11>3.0.CO;2-#
Machaty, Z., Ramsoondar, J. J., Bonk, A. J., Bondioli, K. R., and Prather, R. S. (2002a). Capacitative calcium entry mechanism in porcine oocytes. Biol. Reprod. 66, 667–674. doi:10.1095/biolreprod66.3.667
Machaty, Z., Ramsoondar, J. J., Bonk, A. J., Prather, R. S., and Bondioli, K. R. (2002b). Na+/Ca2+ exchanger in porcine oocytes. Biol. Reprod. 67, 1133–1139. doi:10.1095/biolreprod67.4.1133
Madgwick, S., Hansen, D. V., Levasseur, M., Jackson, P. K., and Jones, K. T. (2006). Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis. J. Cell Biol. 174, 791–801. doi:10.1083/jcb.200604140
Madgwick, S., and Jones, K. T. (2007). How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals Cytostatic Factor. Cell. Div. 2, 4. doi:10.1186/1747-1028-2-4
Mak, D. O., McBride, S., and Foskett, J. K. (1988). Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition. Proc. Natl. Acad. Sci. U. S. A. 95, 15821–15825. doi:10.1073/pnas.95.26.15821
Mak, D. O., McBride, S., and Foskett, J. K. (1999). ATP regulation of type 1 inositol 1,4,5- trisphosphate receptor channel gating by allosteric tuning of Ca2+ activation. J. Biol. Chem. 274, 22231–22237. doi:10.1074/jbc.274.32.22231
Mandadi, S., Armati, P. J., and Roufogalis, B. D. (2011). Protein kinase C modulation of thermo-sensitive transient receptor potential channels: implications for pain signaling. J. Nat. Sci. Biol. Med. 2, 13–25. doi:10.4103/0976-9668.82311
Marangos, P., FitzHarris, G., and Carroll, J. (2003). Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei. Development 130, 1461–1472. doi:10.1242/dev.00340
Markoulaki, S., Matson, S., Abbott, A. L., and Ducibella, T. (2003). Oscillatory CaMKII activity in mouse egg activation. Dev. Biol. 258, 464–474. doi:10.1016/s0012-1606(03)00133-7
Markoulaki, S., Matson, S., and Ducibella, T. (2004). Fertilization stimulates long-lasting oscillations of CaMKII activity in mouse eggs. Dev. Biol. 272, 15–25. doi:10.1016/j.ydbio.2004.04.008
Martín-Romero, F. J., Ortíz-de-Galisteo, J. R., Lara-Laranjeira, J., Domínguez-Arroyo, J. A., González-Carrera, E., and Alvarez, I. S. (2008). Store-operated calcium entry in human oocytes and sensitivity to oxidative stress. Biol. Reprod. 78, 307–315. doi:10.1095/biolreprod.107.064527
Matson, S., Markoulaki, S., and Ducibella, T. (2006). Antagonists of myosin light chain kinase and of myosin II inhibit specific events of egg activation in fertilized mouse eggs. Biol. Reprod. 74, 169–176. doi:10.1095/biolreprod.105.046409
Matsu-Ura, T., Shirakawa, H., Suzuki, K. G. N., Miyamoto, A., Sugiura, K., Michikawa, T., et al. (2019). Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs. Sci. Rep. 9, 4829. doi:10.1038/s41598-019-40931-w
Mazia, D. (1937). The release of calcium in Arbacia eggs on fertilization. J. Cell. Comp. Physiol. 10, 291–304. doi:10.1002/jcp.1030100304
McCulloh, D. H., and Chambers, E. L. (1992). Fusion of membranes during fertilization. Increases of the sea urchin egg's membrane capacitance and membrane conductance at the site of contact with the sperm. J. Gen. Physiol. 99, 137–175. doi:10.1085/jgp.99.2.137
McGuinness, O. M., Moreton, R. B., Johnson, M. H., and Berridge, M. J. (1996). A direct measurement of increased divalent cation influx in fertilised mouse oocytes. Development 122, 2199–2206. doi:10.1242/dev.122.7.2199
Mehlmann, L. M., Mikoshiba, K., and Kline, D. (1996). Redistribution and increase in cortical inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse oocyte. Dev. Biol. 180, 489–498. doi:10.1006/dbio.1996.0322
Mehlmann, L. M., Terasaki, M., Jaffe, L. A., and Kline, D. (1995). Reorganization of the endoplasmic reticulum during meiotic maturation of the mouse oocyte. Dev. Biol. 170, 607–615. doi:10.1006/dbio.1995.1240
Mehregan, A., Ardestani, G., Akizawa, H., Carvacho, I., and Fissore, R. (2021). Deletion of TRPV3 and CaV3.2 T-type channels in mice undermines fertility and Ca2+ homeostasis in oocytes and eggs. J. Cell Sci. 134, jcs257956. doi:10.1242/jcs.257956
Miao, Y. L., Stein, P., Jefferson, W. N., Padilla-Banks, E., and Williams, C. J. (2012). Calcium influx-mediated signaling is required for complete mouse egg activation. Proc. Natl. Acad. Sci. U. S. A. 109, 4169–4174. doi:10.1073/pnas.1112333109
Miyazaki, S. (1988). Inositol 1,4,5-trisphosphate-induced calcium release and guanine nucleotide-binding protein-mediated periodic calcium rises in golden hamster eggs. J. Cell Biol. 106, 345–353. doi:10.1083/jcb.106.2.345
Miyazaki, S. (1991). Repetitive calcium transients in hamster oocytes. Cell Calcium 12, 205–216. doi:10.1016/0143-4160(91)90021-6
Miyazaki, S., Hashimoto, N., Yoshimoto, Y., Kishimoto, T., Igusa, Y., and Hiramoto, Y. (1986). Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs. Dev. Biol. 118, 259–267. doi:10.1016/0012-1606(86)90093-x
Miyazaki, S., Yuzaki, M., Nakada, K., Shirakawa, H., Nakanishi, S., Nakade, S., et al. (1992). Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs. Science 257, 251–255. doi:10.1126/science.1321497
Moos, J., Xu, Z., Schultz, R. M., and Kopf, G. S. (1996). Regulation of nuclear envelope assembly/disassembly by MAP kinase. Dev. Biol. 175, 358–361. doi:10.1006/dbio.1996.0121
Moreno, R. D., Schatten, G., and Ramalho-Santos, J. (2002). Golgi apparatus dynamics during mouse oocyte in vitro maturation: effect of the membrane trafficking inhibitor brefeldin A. Biol. Reprod. 66, 1259–1266. doi:10.1095/biolreprod66.5.1259
Motta, P. M., Nottola, S. A., Makabe, S., and Heyn, R. (2000). Mitochondrial morphology in human fetal and adult female germ cells. Hum. Reprod. 15 (Suppl. 2), 129–147. doi:10.1093/humrep/15.suppl_2.129
Nagaoka, S. I., Hodges, C. A., Albertini, D. F., and Hunt, P. A. (2011). Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors. Curr. Biol. 21, 651–657. doi:10.1016/j.cub.2011.03.003
Nakada, K., Mizuno, J., Shiraishi, K., Endo, K., and Miyazaki, S. (1995). Initiation, persistence, and cessation of the series of intracellular Ca2+ responses during fertilization of bovine eggs. J. Reprod. Dev. 41, 77–84. doi:10.1262/jrd.41.77
Nakano, Y., Shirakawa, H., Mitsuhashi, N., Kuwabara, Y., and Miyazaki, S. (1997). Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon. Mol. Hum. Reprod. 3, 1087–1093. doi:10.1093/molehr/3.12.1087
Nakayama, S., and Kretsinger, R. H. (1994). Evolution of the EF-hand family of proteins. Annu. Rev. Biophys. Biomol. Struct. 23, 473–507. doi:10.1146/annurev.bb.23.060194.002353
Nalefski, E. A., and Falke, J. J. (1996). The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci. 5, 2375–2390. doi:10.1002/pro.5560051201
Nomikos, M., Blayney, L. M., Larman, M. G., Campbell, K., Rossbach, A., Saunders, C. M., et al. (2005). Role of phospholipase C-zeta domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations. J. Biol. Chem. 280, 31011–31018. doi:10.1074/jbc.M500629200
Nomikos, M., Elgmati, K., Theodoridou, M., Georgilis, A., Gonzalez-Garcia, J. R., Nounesis, G., et al. (2011). Novel regulation of PLCζ activity via its XY-linker. Biochem. J. 438, 427–432. doi:10.1042/BJ20110953
Nomikos, M., Mulgrew-Nesbitt, A., Pallavi, P., Mihalyne, G., Zaitseva, I., Swann, K., et al. (2007). Binding of phosphoinositide-specific phospholipase C-ζ (PLC-ζ) to phospholipid membranes: potential role of an unstructured cluster of basic residues. J. Biol. Chem. 282, 16644–16653. doi:10.1074/jbc.M701072200
Norris, R. P., Freudzon, M., Mehlmann, L. M., Cowan, A. E., Simon, A. M., Paul, D. L., et al. (2008). Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption. Development 135, 3229–3238. doi:10.1242/dev.025494
Norris, R. P., Ratzan, W. J., Freudzon, M., Mehlmann, L. M., Krall, J., Movsesian, M. A., et al. (2009). Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 136, 1869–1878. doi:10.1242/dev.035238
Nozawa, K., Satouh, Y., Fujimoto, T., Oji, A., and Ikawa, M. (2018). Sperm-borne phospholipase C zeta-1 ensures monospermic fertilization in mice. Sci. Rep. 8, 1315. doi:10.1038/s41598-018-19497-6
Oh, J. S., Susor, A., and Conti, M. (2011). Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes. Science 332, 462–465. doi:10.1126/science.1199211
Ozil, J. P., Markoulaki, S., Toth, S., Matson, S., Banrezes, B., Knott, J. G., et al. (2005). Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse. Dev. Biol. 282, 39–54. doi:10.1016/j.ydbio.2005.02.035
Ozil, J. P., Sainte-Beuve, T., and Banrezes, B. (2017). [Mg2+]o/[Ca2+]o determines Ca2+ response at fertilization: tuning of adult phenotype? Reproduction 154, 675–693. doi:10.1530/REP-16-0057
Palermo, G., Joris, H., Devroey, P., and Van Steirteghem, A. C. (1992). Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18. doi:10.1016/0140-6736(92)92425-f
Parrington, J., Brind, S., De Smedt, H., Gangeswaran, R., Lai, F. A., Wojcikiewicz, R., et al. (1998). Expression of inositol 1,4,5-trisphosphate receptors in mouse oocytes and early embryos: the type I isoform is upregulated in oocytes and downregulated after fertilization. Dev. Biol. 203, 451–461. doi:10.1006/dbio.1998.9071
Peres, A. (1986). Calcium current in mouse eggs recorded with the tight-seal, whole-cell voltage-clamp technique. Cell. Biol. Int. Rep. 10, 117–119. doi:10.1016/0309-1651(86)90095-0
Periasamy, M., and Kalyanasundaram, A. (2007). SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve 35, 430–442. doi:10.1002/mus.20745
Peters, J. M. (2006). The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat. Rev. Mol. Cell. Biol. 7, 644–656. doi:10.1038/nrm1988
Petersen, C. C., Berridge, M. J., Borgese, M. F., and Bennett, D. L. (1995). Putative capacitative calcium entry channels: expression of Drosophila trp and evidence for the existence of vertebrate homologues. Biochem. J. 311, 41–44. doi:10.1042/bj3110041
Phillips, S. V., Yu, Y., Rossbach, A., Nomikos, M., Vassilakopoulou, V., Livaniou, E., et al. (2011). Divergent effect of mammalian PLCζ in generating Ca2⁺ oscillations in somatic cells compared with eggs. Biochem. J. 438, 545–553. doi:10.1042/BJ20101581
Politi, A., Gaspers, L. D., Thomas, A. P., and Höfer, T. (2006). Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks. Biophys. J. 90, 3120–3133. doi:10.1529/biophysj.105.072249
Putney, J. W. (1990). Capacitative calcium entry revisited. Cell Calcium 11, 611–624. doi:10.1016/0143-4160(90)90016-n
Ramsey, I. S., Delling, M., and Clapham, D. E. (2006). An introduction to TRP channels. Annu. Rev. Physiol. 68, 619–647. doi:10.1146/annurev.physiol.68.040204.100431
Ridgway, E. B., Gilkey, J. C., and Jaffe, L. F. (1977). Free calcium increases explosively in activating medaka eggs. Proc. Natl. Acad. Sci. U. S. A. 74, 623–627. doi:10.1073/pnas.74.2.623
Rogers, N. T., Hobson, E., Pickering, S., Lai, F. A., Braude, P., and Swann, K. (2004). Phospholipase Czeta causes Ca2+ oscillations and parthenogenetic activation of human oocytes. Reproduction 128, 697–702. doi:10.1530/rep.1.00484
Roos, J., DiGregorio, P. J., Yeromin, A. V., Ohlsen, K., Lioudyno, M., Zhang, S., et al. (2005). STIM1, an essential and conserved component of store-operated Ca2+ channel function. J. Cell Biol. 169, 435–445. doi:10.1083/jcb.200502019
Rusnak, F., and Mertz, P. (2000). Calcineurin: form and function. Physiol. Rev. 80, 1483–1521. doi:10.1152/physrev.2000.80.4.1483
Sanders, J. R., Ashley, B., Moon, A., Woolley, T. E., and Swann, K. (2018). PLCζ Induced Ca2+ oscillations in mouse eggs involve a positive feedback cycle of Ca2+ induced InsP3 formation from cytoplasmic PIP2. Front. Cell. Dev. Biol. 6, 36. doi:10.3389/fcell.2018.00036
Sanders, J. R., and Jones, K. T. (2018). Regulation of the meiotic divisions of mammalian oocytes and eggs. Biochem. Soc. Trans. 46, 797–806. doi:10.1042/BST20170493
Sathananthan, A. H., Ng, S. C., Chia, C. M., Law, H. Y., Edirisinghe, W. R., and Ratnam, S. S. (1985). The origin and distribution of cortical granules in human oocytes with reference to Golgi, nucleolar, and microfilament activity. Ann. N. Y. Acad. Sci. 442, 251–264. doi:10.1111/j.1749-6632.1985.tb37526.x
Sathananthan, A. H., and Trounson, A. O. (2000). Mitochondrial morphology during preimplantational human embryogenesis. Hum. Reprod. 15 (Suppl. 2), 148–159. doi:10.1093/humrep/15.suppl_2.148
Sato, K., Wakai, T., Seita, Y., Takizawa, A., Fissore, R. A., Ito, J., et al. (2013). Molecular characteristics of horse phospholipase C zeta (PLCζ). Anim. Sci. J. 84, 359–368. doi:10.1111/asj.12044
Sato, M. S., Yoshitomo, M., Mohri, T., and Miyazaki, S. (1999). Spatiotemporal analysis of [Ca2+]i rises in mouse eggs after intracytoplasmic sperm injection (ICSI). Cell Calcium 26, 49–58. doi:10.1054/ceca.1999.0053
Satouh, Y., Nozawa, K., Yamagata, K., Fujimoto, T., and Ikawa, M. (2017). Viable offspring after imaging of Ca2+ oscillations and visualization of the cortical reaction in mouse eggs. Biol. Reprod. 96, 563–575. doi:10.1093/biolre/iox002
Satouh, Y., and Sato, K. (2023). Reorganization, specialization, and degradation of oocyte maternal components for early development. Reprod. Med. Biol. 22, e12505. doi:10.1002/rmb2.12505
Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., et al. (2002). PLC zeta: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129, 3533–3544. doi:10.1242/dev.129.15.3533
Schuh, M., and Ellenberg, J. (2007). Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 130, 484–498. doi:10.1016/j.cell.2007.06.025
Sette, C., Paronetto, M. P., Barchi, M., Bevilacqua, A., Geremia, R., and Rossi, P. (2002). Tr-kit- induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase. EMBO J. 21, 5386–5395. doi:10.1093/emboj/cdf553
Shilling, F. M., Carroll, D. J., Muslin, A. J., Escobedo, J. A., Williams, L. T., and Jaffe, L. A. (1994). Evidence for both tyrosine kinase and G-protein-coupled pathways leading to starfish egg activation. Dev. Biol. 162, 590–599. doi:10.1006/dbio.1994.1112
Simerly, C., Nowak, G., de Lanerolle, P., and Schatten, G. (1998). Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos. Mol. Biol. Cell. 9, 2509–2525. doi:10.1091/mbc.9.9.2509
Sneyd, J., Tsaneva-Atanasova, K., Reznikov, V., Bai, Y., Sanderson, M. J., and Yule, D. I. (2006). A method for determining the dependence of calcium oscillations on inositol trisphosphate oscillations. Proc. Natl. Acad. Sci. U. S. A. 103, 1675–1680. doi:10.1073/pnas.0506135103
Solc, P., Kitajima, T. S., Yoshida, S., Brzakova, A., Kaido, M., Baran, V., et al. (2015). Multiple requirements of PLK1 during mouse oocyte maturation. PLoS One 10, e0116783. doi:10.1371/journal.pone.0116783
Sorensen, R. A., and Wassarman, P. M. (1976). Relationship between growth and meiotic maturation of the mouse oocyte. Dev. Biol. 50, 531–536. doi:10.1016/0012-1606(76)90172-x
Stice, S. L., and Robl, J. M. (1990). Activation of mammalian oocytes by a factor obtained from rabbit sperm. Mol. Reprod. Dev. 25, 272–280. doi:10.1002/mrd.1080250309
Stojkovic, M., Machado, S. A., Stojkovic, P., Zakhartchenko, V., Hutzler, P., Gonçalves, P. B., et al. (2001). Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol. Reprod. 64, 904–909. doi:10.1095/biolreprod64.3.904
Storey, A., Elgmati, K., Wang, Y., Knaggs, P., and Swann, K. (2021). The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs. Mol. Hum. Reprod. 27, gaaa086. doi:10.1093/molehr/gaaa086
Stricker, S. A. (1999). Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157–176. doi:10.1006/dbio.1999.9340
Stricker, S. A. (2006). Structural reorganizations of the endoplasmic reticulum during egg maturation and fertilization. Semin. Cell. Dev. Biol. 17, 303–313. doi:10.1016/j.semcdb.2006.02.002
Suh, P. G., Park, J. I., Manzoli, L., Cocco, L., Peak, J. C., Katan, M., et al. (2008). Multiple roles of phosphoinositide-specific phospholipase C isozymes. BMB. Rep. 41, 415–434. doi:10.5483/bmbrep.2008.41.6.415
Suzuki, T., Yoshida, N., Suzuki, E., Okuda, E., and Perry, A. C. (2010). Full-term mouse development by abolishing Zn2+-dependent metaphase II arrest without Ca2+ release. Development 137, 2659–2669. doi:10.1242/dev.049791
Swann, K. (1990). A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs. Development 110, 1295–1302. doi:10.1242/dev.110.4.1295
Swann, K. (1994). Ca2+ oscillations and sensitization of Ca2+ release in unfertilized mouse eggs injected with a sperm factor. Cell Calcium 15, 331–339. doi:10.1016/0143-4160(94)90072-8
Swann, K. (1996). Soluble sperm factors and Ca2+ release in eggs at fertilization. Rev. Reprod. 1, 33–39. doi:10.1530/ror.0.0010033
Swann, K. (2022). Sperm Factors and Egg Activation: PLCzeta as the sperm factor that activates eggs: 20 years on. Reproduction 164, E1–E4. doi:10.1530/REP-22-0148
Swann, K. (2023). Sperm-induced Ca2+ release in mammalian eggs: the roles of PLCζ, InsP3, and ATP. Cells 12, 2809. doi:10.3390/cells12242809
Swann, K., and Lai, F. A. (2013). PLCζ and the initiation of Ca2+ oscillations in fertilizing mammalian eggs. Cell Calcium 53, 55–62. doi:10.1016/j.ceca.2012.11.001
Takahashi, T., Kikuchi, T., Kidokoro, Y., and Shirakawa, H. (2013). Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs. Biochem. Biophys. Res. Commun. 430, 60–65. doi:10.1016/j.bbrc.2012.11.024
Tatemoto, H., and Horiuchi, T. (1995). Requirement for protein synthesis during the onset of meiosis in bovine oocytes and its involvement in the autocatalytic amplification of maturation-promoting factor. Mol. Reprod. Dev. 41, 47–53. doi:10.1002/mrd.1080410108
Tesarik, J., Sousa, M., and Testart, J. (1994). Human oocyte activation after intracytoplasmic sperm injection. Hum. Reprod. 9, 511–518. doi:10.1093/oxfordjournals.humrep.a138537
Thanassoulas, A., Swann, K., Lai, F. A., and Nomikos, M. (2022). Sperm Factors and Egg Activation: the structure and function relationship of sperm PLCZ1. Reproduction 23 (164), F1–F8. doi:10.1530/REP-21-0477
Trimarchi, J. R., Liu, L., Porterfield, D. M., Smith, P. J., and Keefe, D. L. (2000). Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos. Biol. Reprod. 62, 1866–1874. doi:10.1095/biolreprod62.6.1866
Trounson, A. O., Chamley, W. A., Kennedy, J. P., and Tassell, R. (1974). Primordial follicle numbers in ovaries and levels of LH and FSH in pituitaries and plasma of lambs selected for and against multiple births. Aust. J. Biol. Sci. 27, 293–299. doi:10.1071/bi9740293
Tu, H., Wang, Z., Nosyreva, E., De Smedt, H., and Bezprozvanny, I. (2005). Functional characterization of mammalian inositol 1,4,5-trisphosphate receptor isoforms. Biophys. J. 88, 1046–1055. doi:10.1529/biophysj.104.049593
Uh, K., Ryu, J., Zhang, L., Errington, J., Machaty, Z., and Lee, K. (2019). Development of novel oocyte activation approaches using Zn2+ chelators in pigs. Theriogenology 125, 259–267. doi:10.1016/j.theriogenology.2018.11.008
Valencia, C., Pérez, F. A., Matus, C., Felmer, R., and Arias, M. E. (2021). Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs. Biol. Reprod. 104, 1126–1138. doi:10.1093/biolre/ioab019
Van Blerkom, J., Davis, P., and Alexander, S. (2003). Inner mitochondrial membrane potential (DeltaPsim), cytoplasmic ATP content and free Ca2+ levels in metaphase II mouse oocytes. Hum. Reprod. 18, 2429–2440. doi:10.1093/humrep/deg466
Van Blerkom, J., Davis, P. W., and Lee, J. (1995). ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum. Reprod. 10, 415–424. doi:10.1093/oxfordjournals.humrep.a135954
Vig, M., Peinelt, C., Beck, A., Koomoa, D. L., Rabah, D., Koblan-Huberson, M., et al. (2006). CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science 312, 1220–1223. doi:10.1126/science.1127883
Wakai, T., and Fissore, R. A. (2019). Constitutive IP3R1-mediated Ca2+ release reduces Ca2+ store content and stimulates mitochondrial metabolism in mouse GV oocytes. J. Cell. Sci. 132, jcs225441. doi:10.1242/jcs.225441
Wakai, T., Mehregan, A., and Fissore, R. A. (2019). Ca2+ signaling and homeostasis in mammalian oocytes and eggs. Cold Spring Harb. Perspect. Biol. 11, a035162. doi:10.1101/cshperspect.a035162
Wakai, T., Vanderheyden, V., Yoon, S. Y., Cheon, B., Zhang, N., Parys, J. B., et al. (2012). Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation. J. Cell. Physiol. 227, 705–717. doi:10.1002/jcp.22778
Wakai, T., Zhang, N., Vangheluwe, P., and Fissore, R. A. (2013). Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs. J. Cell. Sci. 126 (Pt 24), 5714–5724. doi:10.1242/jcs.136549
Wallace, W. H., and Kelsey, T. W. (2010). Human ovarian reserve from conception to the menopause. PLoS One 5, e8772. doi:10.1371/journal.pone.0008772
Wang, C., Lee, K., Gajdócsi, E., Papp, A. B., and Machaty, Z. (2012). Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes. Dev. Biol. 365, 414–423. doi:10.1016/j.ydbio.2012.03.007
Wang, C., Zhang, L., Jaeger, L. A., and Machaty, Z. (2015). Store-operated Ca2+ entry sustains the fertilization Ca2+ signal in pig eggs. Biol. Reprod. 93, 25. doi:10.1095/biolreprod.114.126151
Wassarman, P. M. (1988). Zona pellucida glycoproteins. Annu. Rev. Biochem. 57, 415–442. doi:10.1146/annurev.bi.57.070188.002215
Wassarman, P. M., and Albertini, D. F. (1994). “The mammalian ovum,” in The physiology of reproduction. Editors E. Knobil,, and J. D. Neill (New York: Raven Press).
Watanabe, Y., and Nurse, P. (1999). Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400, 461–464. doi:10.1038/22774
Whitaker, M., and Irvine, R. F. (1984). Inositol 1,4,5-trisphosphate microinjection activates sea urchin eggs. Nature 312, 636–639. doi:10.1038/312636a0
Wu, A. T., Sutovsky, P., Manandhar, G., Xu, W., Katayama, M., Day, B. N., et al. (2007). PAWP, a sperm-specific WW domain-binding protein, promotes meiotic resumption and pronuclear development during fertilization. J. Biol. Chem. 282, 12164–12175. doi:10.1074/jbc.M609132200
Wu, H., He, C. L., and Fissore, R. A. (1997). Injection of a porcine sperm factor triggers calcium oscillations in mouse oocytes and bovine eggs. Mol. Reprod. Dev. 46, 176–189. doi:10.1002/(SICI)1098-2795(199702)46:2<176::AID-MRD8>3.0.CO;2-N
Wu, M., Wu, C., Song, T., Pan, K., Wang, Y., and Liu, Z. (2023). Structure and transport mechanism of the human calcium pump SPCA1. Cell Res. 33, 533–545. doi:10.1038/s41422-023-00827-x
Yoon, S. Y., and Fissore, R. A. (2007). Release of phospholipase C zeta and [Ca2+]i oscillation-inducing activity during mammalian fertilization. Reproduction 134, 695–704. doi:10.1530/REP-07-0259
Yu, F., Sun, L., and Machaca, K. (2009). Orai1 internalization and STIM1 clustering inhibition modulate SOCE inactivation during meiosis. Proc. Natl. Acad. Sci. U. S. A. 106, 17401–17406. doi:10.1073/pnas.0904651106
Yu, Y., Dumollard, R., Rossbach, A., Lai, F. A., and Swann, K. (2010). Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation. J. Cell. Physiol. 224, 672–680. doi:10.1002/jcp.22171
Yu, Y., Nomikos, M., Theodoridou, M., Nounesis, G., Lai, F. A., and Swann, K. (2012). PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P(2). Mol. Biol. Cell 23, 371–380. doi:10.1091/mbc.E11-08-0687
Yu, Y., Saunders, C. M., Lai, F. A., and Swann, K. (2008). Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta. Hum. Reprod. 23, 365–373. doi:10.1093/humrep/dem350
Zhang, L., Chao, C. H., Jaeger, L. A., Papp, A. B., and Machaty, Z. (2018). Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1. Biol. Reprod. 98, 510–519. doi:10.1093/biolre/ioy016
Zhang, L., and Machaty, Z. (2017). The effect of magnesium on the sperm-induced calcium signal in porcine oocytes. Proc. Soc. Study Reproduction 2017, 109.
Zhang, M., Su, Y. Q., Sugiura, K., Xia, G., and Eppig, J. J. (2010). Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science 330, 366–369. doi:10.1126/science.1193573
Keywords: oocyte, egg, sperm, fertilization, calcium, signal transduction, mammals
Citation: Machaty Z (2024) The signal that stimulates mammalian embryo development. Front. Cell Dev. Biol. 12:1474009. doi: 10.3389/fcell.2024.1474009
Received: 31 July 2024; Accepted: 05 September 2024;
Published: 17 September 2024.
Edited by:
Rafael A. Fissore, University of Massachusetts Amherst, United StatesReviewed by:
Luis Aguila, Montreal University, CanadaSook Young Yoon, CHA University, Republic of Korea
Copyright © 2024 Machaty. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Zoltan Machaty, em1hY2hhdHlAcHVyZHVlLmVkdQ==