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Dynamic Changes to the Inositol 1,4,5-Trisphosphate and Ryanodine Receptors during Maturation of Bovine Oocytes
Abstract
The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyR) have been identified as two ligand-gated calcium channels which play a critical role in mediating calcium release in many different types of cells and tissues. The physiological significance of the two receptors in regulation of intracellular calcium during meiotic maturation and fertilization in the bovine oocyte was evaluated. Metabolic labeling of bovine oocytes by Met-Cys 35S during early and late maturation was followed by immunoprecipitation of both RyR and IP3R using specific antibodies against these two receptors. Results indicate that IP3R is translated throughout the maturation period; in contrast, RyR is only translated during the late maturation period of bovine oocytes. In addition, the experiments reported here investigate the temporal and spatial relationships between these calcium channels and the endoplasmic reticulum (ER) and cortical granules (CG). Immunocytochemistry, fluorescence staining and confocal microscopy were applied at four oocyte developmental stages: the germinal vesicleintact (GV-intact), metaphase I (MI) and metaphase II (MII) stages of maturation and the fertilized egg at 6 h post insemination (hpi). Although oocytes demonstrated some differences in staining patterns and localization, both receptor types showed apparent dynamic changes during meiotic maturation and dramatic decreases in signals after insemination. These results indicate the changes in the number and distribution of IP3R and RyR may account for the increased intracellular calcium responsiveness at fertilization. The IP3R appears to associate with the ER at the sub-vitelline membrane cortex in bovine oocytes. In addition, RyR appears to associate with the CG. In conclusion, although these two receptors may have different functional roles in regulation of calcium release during meiotic maturation and fertilization, it appears that both IP3R and RyR contribute to the significant increase of intracellular calcium during fertilization and activation in the bovine oocyte.
... In mature mammalian oocytes, the ER is distributed in cortical ER clusters, and it is assumed that this particular distribution of the ER acts as a cortical pacemaker responsible for generating Ca 2þ waves at fertilization (Dumollard et al., 2002;Kline et al., 1999). The presence of two types of Ca 2þ channels, InsP 3 Rs and ryanodine receptors (RyR), in the ER membrane of the mammalian oocytes is well documented (Machaty et al., 1997;Mehlmann et al., 1996;Stricker, 1999;Wang et al., 2005;Yue et al., 1995). When InsP 3 Rs or RyRs are activated, they contribute to the release of Ca 2þ from the ER lumen, increasing [Ca 2þ ] i . ...
... Consequently, after activation of bovine oocytes by ionomycin, ryanodine, or fertilization, RyR distribution decreases significantly to undetectable levels (Yue et al., 1998). In a similar manner, InsP 3 R clustering decreases after fertilization, and these receptors diffuse into the central region of the bovine oocyte (Wang et al., 2005). Because a sustained increase of cytosolic Ca 2þ leads to the fragmentation of ER tubules in somatic cells (Subramanian and Meyer, 1997), one possibility that can explain the diffusion of the ER-resident Ca 2þ transporters after fertilization is that the rise of cytosolic Ca 2þ in the oocyte, triggered by the sperm fusion, may induce the loss of InsP 3 R and RyR clustering due to the fragmentation of the cortical network of the ER (Stricker, 2006). ...
... As stated for InsP 3 Rs, RyRs show a very weak and diffuse distribution in GV oocytes, whereas the expression is greater in MI and MII oocytes. RyRs are found mainly in the cortex of MI and MII oocytes and are associated to cortical granules in bovine oocytes from the MI stage through fertilization, suggesting that RyRs may play a key role in cortical granule release (Wang et al., 2005;Yue et al., 1998). The activity of these receptors was studied by means of the injection of ryanodine, which triggered a slight increase of cytosolic Ca 2þ in GV oocytes, and a series of significant Ca 2þ spikes in mature oocytes (Yue et al., 1998). ...
Calcium signaling is essential for many cellular events, including muscle contraction, secretion of hormones and neurotransmitters, and fertilization of oocytes. For the appropriate maturation and fertilization of mammalian oocytes, the influx of extracellular calcium through plasma membrane Ca(2+) channels is required. Although the molecular pathway of the Ca(2+) entry in other cell types has been reported, Ca(2+) channels involved in the regulation of Ca(2+) influx in oocytes have remained unknown for a long time. In this review, we summarize recent findings regarding the occurrence of store-operated calcium entry (SOCE) in mammalian oocytes and the expression and localization profiles of STIM1 and ORAI1, two important proteins that control SOCE. As we discuss here, STIM1, as an endoplasmic reticulum Ca(2+) sensor, and ORAI1, the major plasma Ca(2+) channel involved in SOCE, might help to explain the role of Ca(2+) entry in mammalian oocyte maturation and fertilization.
... Less clear are the effects of ryanodine receptors (RyR), whose contribution to fertilization has been well-recognized only in sea urchin [7]. The sensitivity of IP 3 -induced Ca 2+ release (IICR) increases during maturation [8,9] along with IP 3 R-1 [10,11] increase. In bovine, IP 3 R translate throughout maturation period from the perinuclear region of germinal vescicle (GV) to distinct focal clusters at the cortex of the matured oocyte [11]; in contrast, RyR are only translated during the late maturation period. ...
... The sensitivity of IP 3 -induced Ca 2+ release (IICR) increases during maturation [8,9] along with IP 3 R-1 [10,11] increase. In bovine, IP 3 R translate throughout maturation period from the perinuclear region of germinal vescicle (GV) to distinct focal clusters at the cortex of the matured oocyte [11]; in contrast, RyR are only translated during the late maturation period. In addition, IP 3 R appears to be associated with the ER at the sub-vitelline membrane cortex whereas RyR with the cortical granules [11]. ...
... In bovine, IP 3 R translate throughout maturation period from the perinuclear region of germinal vescicle (GV) to distinct focal clusters at the cortex of the matured oocyte [11]; in contrast, RyR are only translated during the late maturation period. In addition, IP 3 R appears to be associated with the ER at the sub-vitelline membrane cortex whereas RyR with the cortical granules [11]. ...
Ion currents and cytosolic free calcium ([Ca(2+)](i)) elevations are crucial events in triggering the complex machinery involved in both gamete maturation and fertilization. Oocyte maturation is triggered by hormone signaling which causes ion currents and [Ca(2+)](i) increase. Extracellular calcium seems to be required for meiosis progression since: (i) calcium depletion in the maturation medium severely affects oocyte developmental competence; (ii) the activity of plasma membrane L-type Ca(2+) currents decreases during maturation; (iii) the exposure to verapamil, a specific Ca(2+) channel blocker, decreases in vitro maturation efficiency. In spermatozoa, maturation initiates inside the epididymis and ends in the female genital tract. During their journey through the female reproductive tract, sperm undergo a dramatic selection and capacitation achieving fertilization competence. Adhesion to the tubal epithelium extends sperm life through depression of [Ca(2+)](i) until capacitation signals trigger an [Ca(2+)](i) elevation followed by sperm release. At fertilization, egg-sperm interaction evokes well-described transient and almost simultaneous events: i.e., fertilization current, a change in resting potential, and an increase in free [Ca(2+)](i) concentration. These events, termed oocyte activation, are the direct consequence of sperm interaction via either activation of a receptor or entry of a sperm factor. The latter hypothesis has been recently supported by the discovery of PCLzeta, a sperm-specific isozyme triggering a dramatic [Ca(2+)](i) increase via inositol 1,4,5-trisphosphate (IP(3)) production. The course of ion currents and [Ca(2+)](i) transients during maturation and fertilization plays a pivotal role in correct embryo development.
... Lack or absence of IP3R significantly compromises oocyte competence. In addition to IP3R, another Ca 2+ release channel receptor, called ryanodine receptor (RyR) has been proposed to be involved in Ca 2+ release following fertilization in mammals (296)(297)(298). The exact role of this receptor in oocytes is yet to be established; however, its involvement in Ca 2+ release during fertilization has been recently addressed in the frog Bufo arenarum. ...
... The exact role of this receptor in oocytes is yet to be established; however, its involvement in Ca 2+ release during fertilization has been recently addressed in the frog Bufo arenarum. This study demonstrated that caffeine, a well known specific RyR agonist, was able to trigger oocyte activation in a dose-dependent manner, while ruthenium red, a specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine (298). ...
The release of a mature healthy egg for fertilization is the center of the entire reproductive process. From the time of embryonic development till fertilization, the oocyte undergoes several stop-and-go periods. In most animals, oocytes are held in meiotic arrest in prophase I prior to ovulation. The ovulatory luteinizing hormone (LH) surge promotes the resumption of meiosis of the arrested oocytes and their progression through the second meiotic cycle, only to be arrested again at metaphase II until fertilization. This review addresses the underlying mechanisms involved in maintaining the oocyte in meiotic arrest as well as the signaling pathways responsible for releasing it from the arrested phase just prior to ovulation until the completion of meiosis at the time of fertilization.
... Maintenance of suppressed IP3 sensitivity in GV oocytes may be a redundant, fail-safe system that tightly blocks cortical granule exocytosis. In other organisms such as mouse (Mehlmann and Kline, 1994;Xu et al., 2003), hamster (Fujiwara et al., 1993), bovine (Wang et al., 2005) and frog (Terasaki et al., 2001;Boulware and Marchant, 2005), an increase in IP3 sensitivity in the process of meiotic maturation has been reported, yet its molecular mechanism is largely unknown. It is conceivable that non-diffusible factors constitute signaling components that drive cortical maturation in parallel with an increase in IP3 sensitivity. ...
... Although not tested, it would be intriguing to address a question of how long the memory for increased IP3 sensitivity is stored in prophase-arrested immature oocytes. Current models presented for molecular bases of alternation in IP3 sensitivity argue phosphorylation of IP3R (Malathi et al., 2003;Jellerette et al., 2004), modulation of Ca 2+ transport effectors (Machaca, 2004), IP3R redistribution (Kobrinsky et al., 1995;Boulware and Marchant, 2005;Wang et al., 2005) concomitant with ER relocalization, IP3R expression (Xu et al., 2003). From our viewpoint that cortical maturation is likely an irreversible process, an increase in IP3 sensitivity might also be associated with a covalent modification/alternation that occurs concomitantly with cortical maturation. ...
Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.
... It is thought that either the intracytoplasmatic calcium stores or plasma membrane calcium channels are related to the acquisition of oocyte meiotic competence ( Boni et al., 2002). Calcium is released from these stores mainly due to the activation of IP3 receptors (Berridge, 1993) whose cytoplasmatic concentrations increase during maturation ( Mehlmann et al., 1996;Wang et al., 2005). This causes an increase in IP3 receptor sensitivity during oocyte maturation ( Fujiwara et al., 1993;Mehlmann and Kline, 1994) and this parameter may be used for evaluating oocyte maturation and, in particular, cytoplasmic maturation. ...
... In contrast to studies on mice and other lower species, only a few studies have been reported on IP3R1 in oocytes of domestic species. In cattle oocytes, IP3R1 expression and phosphorylation have been shown to have a crucial role in [Ca 2+ ]i oscillations during fertilization (Yue et al. 1995;Malcuit et al. 2005;Wang et al. 2005). Our previous study clearly demonstrated that IP3R1 in pig oocytes also can be phosphorylated by M-phase stage-dependent kinases (Ito et al. 2010) and that the decrease in IP 3R1 expression may be involved in the low fertility of cryopreserved oocytes (Hirose et al. 2013). ...
At fertilization, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) has a crucial role in Ca2+ release in mammals. Expression levels, localization and phosphorylation of IP3R1 are important for its function, but it still remains unclear which molecule(s) regulates IP3R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP3R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP3R1 were formed in the cortex of the oocyte except in a ring-shaped band of cortex adjacent to the spindle. However, no such clusters of IP3R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP3R1 localization, phosphorylation and expression using M-phase stage-dependent kinase inhibitors. Our results show that treatments with roscovitine (p34cdc2 kinase inhibitor) or U0126 (mitogen-activated protein kinase inhibitor) did not affect IP3R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI-2536, an inhibitor of polo-like kinase 1 (Plk1), dramatically decreased the expression level of IP3R1 in pig oocytes in a dose-dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP3R1 expression in pig oocytes.
... In hamster eggs, the spatiotemporal development of the Ca 2+ signal at fertilization is only linked to the InsP 3mediated Ca 2+ release [122], because a feedback system underlying calcium-induced calcium release mediated by RyRs [135] is lacking. Very recently, the RyRs have been shown to directly associate with GC in bovine oocytes (the presence of a ryanodinesensitive calcium release channel has been previously shown to be located within the sea urchin egg cortex as well [136]) from metaphase I stage through fertilization, suggesting that these granules may have a physiological role in the cortical exocytosis process [137]. The second messenger NAADP, in which the nicotinamide ring in the molecule of NADP is replaced by nicotinic acid, has also been found to release Ca 2+ from intact eggs [138]. ...
The beginning of new cell life is dependent on the signaling events that occur inside the sperm and the egg. From the moment when the sperm acquires the ability to swim to the egg to its attachment to the plasma membrane of the latter, Ca 2+ acts as the key messenger that allows essential information exchange between the two cells. A large body of evidence is now available on the importance of Ca 2+ in the activation of sperm prior to the fertilization events. On the egg side, the signaling pathways that trigger the Ca 2+ release at fertilization have been extensively studied for at least 30 years. Even if several lines of evidence indicate that the increase of Ca 2+ in fertilized mammalian eggs demands the activation of inositol 1,4,5-trisphosphate receptors (InsP 3 Rs), the molecular mechanisms responsible for the Ca 2+ increase in the fertilization of other animal species have not been conclusively established.
... Two types of intracellular calcium channels, located in specialized regions of the endoplasmic reticulum (ER), have been identified to control calcium signalling in eggs: the universal inositol trisphosphate receptor (IP 3 R), and the species-specific ryanodine receptor (RyR), which can be found individually or together depending on the particular egg species (Galione et al., 1991(Galione et al., , 1993aMcPherson et al., 1992;Swann, 1992;Nuccitelli et al., 1993;Fissore & Robl, 1993;Lee et al., 1993;Kline & Kline, 1994;Ayabe et al., 1995;Yue et al., 1995Yue et al., , 1998Sousa et al., 1996;Albrieux et al., 1997;Herbert et al., 1997;Macháty et al., 1997;Balakier et al., 2002;Petr et al., 2002;Tesarik, 2002;Wang et al., 2005). Our laboratory has previously reported evidence about the existence and functionality of IP 3 Rs and RyRs of in vitro matured Bufo arenarum oocytes. ...
SummaryTransient increases in the concentration of cytosolic Ca2+ are essential for triggering egg activation events. Increased Ca2+ results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A2 (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A2 (PLA2) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA2 activator, and arachidonic acid, the main PLA2 reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.
... Other notable oocyte proteins with a connection to UPP have been identified in our ExacTag ™ trial: RyR, upregulated in the high-quality oocytes, is involved in calcium signaling during fertilization and has been shown to be translated only during the final stages of bovine oocyte maturation [45]. The number of RyR receptors is thought to be regulated by UPP in skeletal muscle [46]. ...
Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals.
PerkinElmer ExacTag™ Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte-conditioned in vitro maturation media (oocyte secretome) obtained with high- and low-quality oocytes.
We identified 16 major proteins in the oocyte proteome that were expressed differentially in high- versus low-quality oocytes. More abundant proteins in the high-quality oocyte proteome included kelch-like ECH-associated protein 1 (an adaptor for ubiquitin-ligase CUL3), nuclear export factor CRM1 and ataxia-telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low-quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low-quality-oocyte secretome.
A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non-invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.
... It has been established that independent IP 3 Rs and RyRs coexist in mature bovine oocytes (Yue et al., 1995). The ryanodine receptor was observed uniformly localized in the periphery of mature oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes (Yue et al., 1998) Although these two receptors may have different functional roles in the regulation of calcium release during meiotic maturation and fertilization, it appears that both IP 3 Rs and RyRs contribute to the significant increase in intracellular calcium during fertilization and activation in the bovine oocyte (Wang et al., 2005). ...
Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.
... An increase in the sensitivity to IP 3 during oocyte maturation likely involves several modifications in the oocyte during maturation, including a reorganization of Ca 2þ stores as well as increased numbers of IP 3 receptors [8, 9]. Indeed, the ER undergoes a dramatic reorganization during maturation of a diverse array of species (from marine worms to starfish, frogs, rodents, and cows10111213141516) as well as an increase in the amount of IP 3 receptor protein [8, 17]. In GV-stage mouse oocytes, the ER is continuous with the nuclear envelope and is present throughout the cytoplasm as well as in small accumulations throughout the oocyte interior. ...
Oocyte maturation in rodents is characterized by a dramatic reorganization of the endoplasmic reticulum (ER) and an increase in the ability of an oocyte to release Ca(2+) in response to fertilization or inositol 1,4,5-trisphosphate (IP(3)). We examined if human oocytes undergo similar changes during cytoplasmic meiotic maturation both in vivo and in vitro. Immature, germinal vesicle (GV)-stage oocytes had a fine network of ER throughout the cortex and interior, whereas the ER in the in vivo-matured, metaphase II oocytes was organized in large (diameter, ∼2-3 μm) accumulations throughout the cortex and interior. Likewise, oocytes matured in vitro exhibited cortical and interior clusters with no apparent polarity in regard to the meiotic spindle. In vivo-matured oocytes contained approximately 1.5-fold the amount of IP(3) receptor protein and released significantly more Ca(2+) in response to IP(3) compared with GV-stage oocytes; however, oocytes matured in vitro did not contain more IP(3) receptor protein or release more Ca(2+) in response to IP(3) compared with GV-stage oocytes. These results show that at least one cytoplasmic change occurs during in vitro maturation of human oocytes that might be important for fertilization and subsequent embryonic development, but they suggest that a low developmental competence of in vitro-matured oocytes could be the result of deficiencies in the ability to release Ca(2+) at fertilization.
... It has been reported that cell cycle progression occurs synchronously with spontaneous Ca 2+ events [17,65,66]. These events can be [Ca 2+ ] i within a single cell during superfusion with the IP 3 R antagonist 2-aminoethoxydiphenyl borate (2-APB, 10 μM), or with Ryanodine (Rya InhC, 10 μM), which at this concentration acts as a RyR antagonist. ...
Spontaneous Ca(2+) events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca(2+) transients during the G(1) to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP(3)Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP(3)R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP(3)R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP(3)Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.
... First, we show that endogenous STIM1 is expressed in oocytes, and that this expression is remarkably dependent on the maturation stage, being upregulated after GVBD. This developmentally upregulated profile of protein expression is similar to that found for other Ca 2C transport systems that have been shown to be involved in the generation of calcium waves at fertilization in oocytes, such as InsP 3 R and RyR (He et al. 1997, Parrington et al. 1998, Wang et al. 2005). Therefore, the SOCE, a STIM1-dependent pathway, could be considered to be a probable contributor in Ca 2C signaling in oocytes. ...
Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization.
... However, the presence of ryanodine-sensitive stores has been detected in oocytes of several mammalian species (Swann, 1992; Molecular Reproduction and Development. DOI 10.1002/mrd Author Proof A Yue et al., 1995;Wang et al., 2005) including the human (Sousa et al., 1996), in which the ryanodine-sensitive stores were observed to be abundant in the cytoplasm with the exception of the cortical and subcortical regions where the InsP 3 -stores prevail. This last study however deals with data obtained with mature human oocytes, i.e. at a further stage of meiosis, where the nucleus no longer exists. ...
Our aim was to investigate if human oocytes, like mouse oocytes, exhibit spontaneous Ca(2+) oscillations and nuclear translocation of PLC-beta1 prior to germinal vesicle breakdown (GVBD), and to correlate these events with the evolution of chromatin configuration as a landmark for the meiosis resumption kinetics. Human germinal vesicle (GV) oocytes were either loaded with Fluo-3 probe to record Ca(2+) signals or fixed for subsequent fluorescent labeling of both chromatin and PLC-beta1, and immunogold labeling of PLC-beta1. Here for the first time, we show that human oocytes at the GV-stage exhibit spontaneous Ca(2+) oscillations. Interestingly, only oocytes with a large diameter and characterized by a compact chromatin surrounding the nucleolus of the GV could reveal these kind of oscillations. We also observed a translocation of PLC-beta1 from the cytoplasm towards the nucleus during in vitro maturation of human oocytes. Spontaneous calcium oscillations and nuclear translocation of PLC-beta1 may reflect some degree of oocyte maturity. The impact of our results may be very helpful to understand and resolve many enigmatic problems usually encountered during the in vitro meiotic maturation of human GV oocytes.
Fertilization‐induced [Ca2+]i oscillations generally depend on the release of calcium ions from the endoplasmic reticulum (ER). Since ER is the main store of calcium ions, it plays an important role in oocyte fertilization. However, the mechanism of ER organization at oocyte activation is unknown. Here, we show that protein kinase C (PKC) is involved in ER distribution during bovine oocyte activation, but not involved in cell cycle resumption and spindle organization. Actin filaments were affected by PKC pharmacological inhibition. In addition, similar to PKC results, the actin‐depolymerizing drug cytochalasin B affected the ER distribution during oocyte activation. Specifically, we have demonstrated that ER organization during bovine oocyte activation is regulated by PKC possibly through its action on actin filaments regulation. Taken together, the results presented here provide further information on the pathway involved in the regulation of ER organization during oocyte activation and new insight into the functional role of PKC and actin filaments during this process. Inhibition of protein kinase C (PKC) and actin filaments polymerization affects endoplasmic reticulum (ER) reorganization during bovine oocyte parthenogenetic activation.
Summary Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 μM was able to induce 95% activation, while 2H-DhL 36 μM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.
Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca(2+) oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1-2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.
Consequences of heat stress exposure during the first 12 h of meiotic maturation differed depending on how and when bovine oocytes were activated. If heat-stressed oocytes underwent IVF at ~24 h, blastocyst development was less than for respective controls and similar to that obtained for nonheat-stressed oocytes undergoing IVF at 30 h (i.e. slightly aged). In contrast, if heat-stressed oocytes underwent chemical activation with ionomycin/6-dimethylaminopurine at 24 h, blastocyst development was not only higher than respective controls, but also equivalent to development obtained after activation of nonheat-stressed oocytes at 30 h. Developmental differences in chemically activated vs IVF-derived embryos were not related to fertilization failure or gross alterations in cytoskeletal components. Rather, ionomycin-induced calcium release and MAP kinase activity were less in heat-stressed oocytes. While underlying mechanisms are multifactorial, ability to obtain equivalent or higher development after parthenogenetic activation demonstrates that oocytes experiencing heat stress during the first 12 h of meiotic maturation have the necessary components to develop to the blastocyst stage, but fail to do so after fertilization.
An arginine-glycine-aspartic acid (RGD)-containing peptide has been reported to generate calcium transients in bovine oocytes similar to those observed at fertilization. The research objective herein was to evaluate the response of bovine oocytes to an RGD peptide after injection with known antagonists of calcium releasing mechanisms in order to determine the initial calcium releasing pathway. Oocytes were injected with either heparin, an inhibitor of inositol 1,4,5-trisphosphate (IP3) induced calcium response, or procaine, which inhibits calcium release through the ryanodine receptor. Oocytes injected with heparin prior to RGD exposure did not display a calcium response. Oocytes injected with procaine prior to RGD exposure did exhibit a calcium response. Electroporation of IP3, caffeine, or exposure to RGD alone elicited a calcium response for each treatment group. Injection of heparin, procaine, vehicle medium (VM), or exposure to a non-RGD-containing peptide alone failed to elicit a calcium response. The data indicates that the RGD peptide is able to induce calcium transients in oocytes inhibited with procaine, but not those inhibited with heparin. These data suggest the pathway whereby the RGD peptide induces the first intracellular calcium transient in bovine oocytes is through IP3-mediated stores.
The analysis of differences between juvenile and adult oocytes may provide useful information on the acquisition of meiotic and developmental competence of the female gamete. In oocytes collected from either ewes or 40-day-old lambs, we evaluated membrane electrical properties, such as resting potential, conductance, activation ion currents, L-type Ca(2+) currents as well as calcium stores and IP3 sensitivity; in addition, the incidence of apoptosis in cumulus cells in these two age categories was compared. The analysis was carried out in oocytes both prior to and after in vitro maturation. Significant differences were found in all the examined parameters in relation to maturational stages whereas minor differences were recorded in relation to age of the donor. IP3 sensitivity strongly increased after in vitro maturation following a dose-dependent pattern from 1 to 500 micromol/L with a significant interaction (P < 0.01) between dose and maturational stage. The incidence of apoptosis in cumulus cells strongly increased after in vitro maturation and was greater in adult than in juvenile cumulus cells (39.2 +/- 5.8% vs. 21.9 +/- 3.5%; P < 0.01). In conclusion, all the examined parameters were greatly affected by the maturational stage, whereas minor differences were due to age-related oocyte quality, that is, at plasma membrane levels to conductance, activation current peaks and calcium currents, at cytosol level to calcium stores and IP3 sensitivity, and to incidence of apoptosis in cumulus cells. These parameters were compared with previous data in bovine to analyze oocyte quality in juvenile and adult individuals or between species.
The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca²⁺ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [³H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of ∼ 30 and ∼ 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron εscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr ∼ 400,000 peptides/30 S ryanodine receptor oligomer. [³H]Ryanodine binding to the membrane-bound receptor in 50 εM-−1 mM free Ca²⁺ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH ∼ 0.45) sites with a ratio of 1:3. Reduction in free Ca²⁺ to less than or equal to 0.1 εM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca²⁺ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [³H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca²⁺ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr ∼ 400,000.
Fertilization of mature mouse oocytes triggered highly repetitive Ca2+ oscillations lasting 2-3 h. However, immature oocytes generated only two or three oscillations, which ceased within 1 h.
Development of repetitive Ca2+ transients to sperm occurred late in oocyte maturation and was dependent on cytoplasmic modifications that were independent
of cell cycle progression from metaphase I to metaphase II. Immature oocytes released significantly less Ca2+ from stores than mature oocytes in response to ionomycin and thapsigargin. Ryanodine had no effect on intracellular Ca2+ in maturing oocytes but stimulated an increase in Ca2+ in mature oocytes. The ability of ryanodine to increase Ca2+ levels was, however, strain-dependent.
Preincubation of oocytes with thapsigargin or ryanodine significantly attenuated the normal fertilization Ca2+ response, causing a decrease in the number and the rate of rise of the transients. The inhibition of sperm-induced Ca2+ transients by ryanodine was independent of its ability to cause an immediate Ca2+ increase. Low concentrations of ryanodine had no effect on resting Ca2+ levels but inhibited Ca2+ oscillations at fertilization. Similarly Ca2+ oscillations were blocked in oocytes from a strain of mouse that showed no immediate Ca2+ increase with ryanodine. These results suggest that modifications in Ca2+ stores and ryanodine-sensitive Ca2+ release mechanisms during oocyte maturation play an important role in Ca2+ oscillations at fertilization.
At fertilization, the spermatozoon is generally held to generate two important second messengers, inositol trisphosphate and diacylglycerol. A similar situation arises when these signalling molecules are generated after a hormone binds to its plasma membrane receptor. This signalling mechanism releases intracellular Ca2+ which causes cortical granule release and initiates meiotic resumption. This review will examine the role played at fertilization by protein kinase C which is a primary target of diacylglycerol. The pharmacological agents phorbol esters, which mimic the action of diacylglycerol, when added to mammalian oocytes induce cortical granule release and may cause meiotic resumption. However, the originally accepted mechanism of fertilization is now questioned with the recent finding of a soluble sperm Ca2+-releasing factor expelled directly into the oocyte cytoplasm, bypassing any membrane receptor. Therefore, it is timely to re-evaluate the role played by protein kinase C at fertilization in light of a mechanism that may produce Ca2+ without producing diacylglycerol concomitantly. This article will examine the evidence implicating activation of protein kinase C in Ca2+ oscillations, cortical granule release and meiotic resumption. It will contend that pharmacological studies relying on the specificity of phorbol esters and other agonists, as well as inhibitors of protein kinase C, have produced conflicting interpretations of the role of this kinase at fertilization.
We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs. Immunolocalization of the calcium release channel reveals a cortical reticulum or honeycomb staining network that surrounds cortical granules and is associated with the plasma membrane. The network consists of some immunoreactive electron-dense material coating small vesicles and elongate cisternae of the endoplasmic reticulum. The fluorescent reticular staining pattern is lost when egg cortices are treated with agents known to affect sarcoplasmic reticulum calcium release and induce cortical granule exocytosis (ryanodine, calcium, A-23187, and caffeine). An approximately 380-kD protein of sea urchin egg cortices is identified by immunoblot analysis with the ryanodine receptor antibody. These results demonstrate: (a) the presence of a ryanodine-sensitive calcium release channel that is located within the sea urchin egg cortex; (b) an altered calcium release channel staining pattern as a result of treatments that initiate the cortical granule reaction; and (c) a spatial and functional dichotomy of the ER which may be important in serving different roles in the mobilization of calcium at fertilization.
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.
Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.
A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.
Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.
The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.
The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.
Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.
The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.
Inositol 1,4,5-trisphosphate is a second messenger of the phosphoinositide system which can mobilize calcium from intracellular stores. Rat cerebellum is an abundant source of a receptor for inositol 1,4,5-trisphosphate (Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136). In this study we have solubilized and purified this receptor to apparent homogeneity from rat cerebellum. Crude membrane, detergent-solubilized, and purified receptor preparations display similar selectivity for inositol 1,4,5-trisphosphate over other inositol phosphates. The purified receptor is globular with a Stokes' radius of approximately 10 nm. Electrophoretic analysis reveals one protein band with an Mr of 260,000. While binding is reversibly inhibited by 300 nM calcium in particulate fractions and detergent-solubilized membranes, the purified protein is not inhibited by calcium concentrations up to 1.5 mM. Inhibition by calcium is reconstituted by addition of detergent-solubilized cerebellar membranes, but not by the cytosolic fraction of cerebellum.
Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.
Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.
The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we show that highly purified preparations of IP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the IP3R-FKBP12 interaction increases Ca2+ flux through IP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to IP3R, regulating its Ca2+ conductance.
Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50-250 nM IP3 or 10-20 mM caffeine, 100-200 microM ryanodine and 4-8 microM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 microM. Prior injection of either 50 microM ruthenium red, 50 microM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10-20 mM caffeine or 200 microM ryanodine, but prior injection of 50 microM ruthenium red or procaine completely inhibited the effect of 10-20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 microM concentration of ruthenium red or procaine only partially blocked the effect of 200 microM ryanodine, but 200 microM ruthenium red or procaine completely blocked the effect of 200 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)
Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4-5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.
Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.
Calcium oscillations occur during meiotic maturation of mouse oocytes. They also trigger activation at fertilization. We have monitored [Ca2+]i in oocytes at different stages of growth and maturation to examine how the calcium release mechanisms alter during oogenesis. Spontaneous calcium oscillations occur every 2-3 minutes in the majority of fully grown (but immature) mouse oocytes released from antral follicles and resuming meiosis. The oscillations last for 2-4 hours after release from the follicle and take the form of global synchronous [Ca2+]i increases throughout the cell. Rapid image acquisition or cooling the bath temperature from 28 degrees C to 16 degrees C did not reveal any wave-like spatial heterogeneity in the [Ca2+]i signal. Calcium appears to reach highest levels in the germinal vesicle but this apparent difference of [Ca2+] in nucleus and cytoplasm is an artifact of dye loading. Smaller, growing immature oocytes are less competent: about 40% are able to resume meiosis and a similar proportion of these oocytes show spontaneous calcium oscillations. [Ca2+]i transients are not seen in oocytes that do not resume meiosis spontaneously in vitro. Nonetheless, these oocytes are capable of [Ca2+]i oscillations since they show them in response to the addition of carbachol or thimerosal. To examine how the properties of calcium release change during meiotic maturation, a calcium-releasing factor from sperm was microinjected into fully grown immature and mature oocytes. The sperm-factor-induced oscillations were about two-fold larger and longer in mature oocytes compared to immature oocytes. Calcium waves travelling at 40-60 microns/second were generated in mature oocytes, but not in immature oocytes. In some mature oocytes, successive calcium waves had different sites of origin. The modifications in the size and spatial organization of calcium transients during oocyte maturation may be a necessary prerequisite for normal fertilization.
Fertilization of mature mouse oocytes triggered highly repetitive Ca2+ oscillations lasting 2-3 h. However, immature oocytes generated only two or three oscillations, which ceased within 1 h. Development of repetitive Ca2+ transients to sperm occurred late in oocyte maturation and was dependent on cytoplasmic modifications that were independent of cell cycle progression from metaphase I to metaphase II. Immature oocytes released significantly less Ca2+ from stores than mature oocytes in response to ionomycin and thapsigargin. Ryanodine had no effect on intracellular Ca2+ in maturing oocytes but stimulated an increase in Ca2+ in mature oocytes. The ability of ryanodine to increase Ca2+ levels was, however, strain-dependent. Preincubation of oocytes with thapsigargin or ryanodine significantly attenuated the normal fertilization Ca2+ response, causing a decrease in the number and the rate of rise of the transients. The inhibition of sperm-induced Ca2+ transients by ryanodine was independent of its ability to cause an immediate Ca2+ increase. Low concentrations of ryanodine had no effect on resting Ca2+ levels but inhibited Ca2+ oscillations at fertilization. Similarly Ca2+ oscillations were blocked in oocytes from a strain of mouse that showed no immediate Ca2+ increase with ryanodine. These results suggest that modifications in Ca2+ stores and ryanodine-sensitive Ca2+ release mechanisms during oocyte maturation play an important role in Ca2+ oscillations at fertilization.
We established a novel method to isolate a single type of inositol 1,4,5-trisphosphate receptor (IP3R) among the heterogeneous population of receptors to study the regulatory mechanism of Ca2+ release. We raised in the rabbit a polyclonal antibody against synthetic peptide corresponding to amino acids 2736-2747 (pep 6) of type I IP3R (IP3-R-I) that is most abundant in cerebellum. We purified IP3R-I from a 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid solubilized mouse cerebellar microsomal fraction by immunoaffinity chromatography on an anti-pep 6 antibody-Sepharose 4B column with specific elution by the pep 6 peptide (GHPPHMNVNPQQ) of the IP3R-I C terminus. Immunoaffinity-purified IP3R reconstituted into lipid vesicles formed a homotetramer structure. Monoclonal antibody 18A10, which partially blocks the Ca2+ release from cerebellar microsome, almost completely inhibited IP3-induced 45Ca2+ influx into proteoliposomes, whereas monoclonal antibody that recognizes other regions did not inhibit Ca2+ influx. Both the rate and extent of 45Ca2+ influx into proteoliposomes increased 20% after incubation with the catalytic subunit of cyclic AMP-dependent protein kinase, accompanied by stoichiometric phosphorylation of IP3R protein.
Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, or lysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (IP3) to these internal vesicles reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of 1.6 +/- 0.3 nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysis of first-strand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IP3 receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form) and not the long form (neuronal form) detected in rat brain tissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and the IP3 receptor with a Kd of 0.2 nM. Most importantly, the binding of ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.
At fertilization of the mammalian egg, resumption of the cell cycle and the cortical reaction are two events of egg activation, correlated with an increase in intracellular Ca 2+ concentration and activation of protein kinase C. To evaluate the pathways leading to both events, rat eggs were parthenogenetically activated by the calcium ionophore ionomycin, or by the protein kinase C activators 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG). Cortical granule exudate was visualized by the lectin Lens culinaris and Texas Red streptavidin, using a confocal microscope. Resumption of meiosis was detected by Hoechst dye, and intracellular Ca 2+ concentration by fura-2. lonomycin triggered both a cortical reaction and resumption of meiosis, while chelation of intracellular Ca 2+ rise by BAPTA-AM (1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester) revealed a segregation between these two events. A low Ca 2+ transient (∼ 150 nM) induced a partial cortical reaction in half of the eggs, but the meiotic status was not affected. TPA triggered a cortical reaction with neither resumption of meiosis nor intracellular Ca 2+ rise, while OAG induced both aspects of activation, as well as a significant intracellular Ca 2+ rise. We conclude that in the cascade of events leading to egg activation, the initial Ca 2+ rise is followed by a segregation in the pathway. A relatively low Ca 2+ rise is sufficient to induce a partial cortical reaction. However, a higher level of Ca 2+ is required to complete the cortical reaction and resumption of meiosis. The activation of the cell cycle is Ca 2+ -dependent, but protein kinase C-independent.
We have previously reported that injection of ryanodine receptor agonists into mature bovine oocytes induces intracellular calcium release, indicative of the existence of ryanodine receptors. In this experiment, further evidence of the ryanodine receptor localization, and developmental regulation in bovine oocytes is presented. The possible physiological significance is also suggested. Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes. As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more intense and continuous, yet not comparable to that observed in the metaphase II oocytes. These modifications correlate with the intracellular calcium responsiveness to ryanodine. A 200-microM injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stage bovine oocytes, while peaked intracellular calcium transients are subsequently observed in the metaphase II-stage oocytes. However, no significant changes in the amplitude of intracellular calcium transients induced by 250 nM inositol 1,4,5-trisphosphate and 10 microM ionomycin were observed in oocytes at a comparable stage. Fertilization induced a significant decrease in ryanodine receptor signal; similar changes were also observed in oocytes injected with 200 microM ryanodine or incubated with 10 microM ionomycin. However, no changes in ryanodine signal were observed in oocytes injected with vehicle medium. Furthermore, injection of either ryanodine or inositol 1,4,5-trisphosphate induced subsequent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cellular changes following fertilization; stimulation of this receptor results in the activation of bovine oocytes, and it is likely that this receptor may play a role at fertilization.
The presence of different intracellular Ca2+ release mechanisms in porcine oocytes and their involvement in mediating Ca2+ transients in different developmental stages were investigated. Metaphase II arrested oocytes showed an increase in intracellular Ca2+ concentration after injection of inositol 1,4,5-trisphosphate (InsP3), the InsP3 receptor agonist. Similar Ca2+ spikes could be detected after injection of ryanodine and cyclic ADP ribose, the ryanodine receptor agonists. The InsP3-induced Ca2+ release was inhibited by heparin, the InsP3 receptor antagonist, whereas procaine, the ryanodine receptor antagonist, blocked the Ca2+ transients generated by ryanodine and cyclic ADP ribose. In germinal vesicle-stage oocytes, intracellularly stored Ca2+ could also be mobilized by agonist treatment, though the effective concentration to generate the Ca2+ spikes was higher. After in vitro fertilization, repetitive Ca2+ transients were generated in oocytes starting 2.5-3 h after insemination. They ceased around the time of pronuclear formation when the oocytes entered first interphase. At this stage, the receptors were still capable of mediating Ca2+ release upon agonist treatment; in many cases these spikes were of longer duration, suggesting that in interphase it takes a longer time for the Ca2+ stores to resequester the mobilized Ca2+ from the cytosol. These results suggest that porcine oocytes possess both InsP3 and ryanodine Ca2+ channel receptors and that the properties of the Ca2+ release mechanisms change during oocyte development.
During maturation, mammalian oocytes undergo a series of changes that prepare them for fertilization. These events are regulated by kinases, most notably histone H1 and mitogen-activated protein kinase. Intracellular calcium ([Ca2+]i) oscillations participate in oocyte signaling, and it has been postulated that they play a role in oocyte maturation. In these studies we investigated the association of Ca2+, Ca2+ channels, and activation of kinases in in vitro-maturating bovine oocytes. BAPTA-AM, a Ca2+ chelator, inhibited oocyte maturation and delayed activation of kinases, although spontaneous [Ca2+]i rises were not observed in control oocytes loaded with fura-2, a Ca2+ indicator. The ability of the 1,4,5-inositol trisphosphate receptor (InsP3R) to release Ca2+, monitored after the addition of thimerosal and myo-inositol 1,4,5-trisphosphate (InsP3), increased as maturation progressed. This may be associated with a similar increase, monitored by Western blotting, in the density of the type I InsP3R isoform during oocyte maturation. Injection of heparin, an InsP3R antagonist, blocked oocyte maturation and activation of kinases. The density of the ryanodine receptor, another Ca2+ channel, may be 30- to 100-fold lower than that of the InsP3R in bovine oocytes. Thus, our results demonstrate that [Ca2+]i participates in the progression of meiosis and that the InsP3R may be responsible for the majority of Ca2+ release during maturation and fertilization.
We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs. Immunolocalization of the calcium release channel reveals a cortical reticulum or honeycomb staining network that surrounds cortical granules and is associated with the plasma membrane. The network consists of some immunoreactive electron-dense material coating small vesicles and elongate cisternae of the endoplasmic reticulum. The fluorescent reticular staining pattern is lost when egg cortices are treated with agents known to affect sarcoplasmic reticulum calcium release and induce cortical granule exocytosis (ryanodine, calcium, A-23187, and caffeine). An approximately 380-kD protein of sea urchin egg cortices is identified by immunoblot analysis with the ryanodine receptor antibody. These results demonstrate: (a) the presence of a ryanodine-sensitive calcium release channel that is located within the sea urchin egg cortex; (b) an altered calcium release channel staining pattern as a result of treatments that initiate the cortical granule reaction; and (c) a spatial and functional dichotomy of the ER which may be important in serving different roles in the mobilization of calcium at fertilization.
A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.
The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.
The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.
A computer-assisted sequence analysis of the ryanodine receptor pointed to a 15-residue peptide, "KC7", reported to have been purified from a proteolytic digest of the 565 kDa rabbit skeletal muscle protein. Sequence comparisons, however, showed that this peptide probably originated from a much smaller protein which copurified with the ryanodine receptor. Peptide KC7 (excluding its unknown N-terminal residue) was identical to the N-terminus of a 12 kDa immunophilin (immunosupressant-binding protein), human T-cell FK506- binding protein (FKBP), which has recently been identified as an inhibitor of protein kinase C. There was no other sequence similarity between FKBP and the ryanodine receptor. It is suggested that in vivo interaction of the ryanodine receptor and FKBP may play a role in the modulation of calcium release in muscle.
The calcium release channel from rabbit muscle sarcoplasmic reticulum (SR) has been purified and reconstituted as a functional unit in lipid bilayers. Electron microscopy reveals the four-leaf clover structure previously described for the 'feet' that span the transverse tubule (T)-SR junction. Ca2+ release from the SR induced by T-system depolarization during excitation-contraction coupling in muscle may thus be effected through a direct association of the T-system with SR Ca2+-release channels.
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.
Cardiac ryanodine receptor was purified from canine ventricle as a single polypeptide of Mr 400,000 by a stepwise sucrose density gradient centrifugation and heparin-Sepharose CL-4B column chromatography. The [3H]ryanodine binding capacity (Bmax) was 60-fold enriched from cardiac microsomes without a change in affinity for [3H]ryanodine. The purity of the final preparation was determined to be greater than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified preparation as an antigen, we produced six monoclonal antibodies which immunoprecipitated the cardiac ryanodine receptor. Three of these antibodies recognized the cardiac receptor on immunoblot analysis. In contrast, no protein in the microsomes isolated from Type I (slow) or Type II (fast) skeletal muscles was recognized by these antibodies. The [3H]ryanodine binding to cardiac and skeletal muscle microsomes was dependent on free Ca2+ concentration. In skeletal muscle microsomes, the [3H]ryanodine binding was remarkably enhanced by the addition of ATP or KCl and inhibited by high free Ca2+, whereas it was less sensitive to these agents in cardiac microsomes. All of these results clearly demonstrate that the cardiac ryanodine receptor is different from the skeletal muscle receptors and is not present even in Type I (slow) skeletal muscle fibers, in which cardiac isoforms of some of the muscle proteins are constitutively expressed.
The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
FK506-binding protein (FKBP12) was originally identified as the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. The cellular function of FKBP12, a ubiquitously expressed 12,000-dalton proline isomerase, has been unknown. FKBP12 copurifies with the 565,000-dalton ryanodine receptor (RyR), four of which form intracellular Ca2+ release channels of the sarcoplasmic and endoplasmic reticula. By coexpressing the RyR and FKBP12 in insect cells, we have demonstrated that FKBP12 modulates channel gating by increasing channels with full conductance levels (by > 400%), decreasing open probability after caffeine activation (from 0.63 +/- 0.09 to 0.04 +/- 0.02), and increasing mean open time (from 4.4 +/- 0.6 ms to 75 +/- 41 ms). FK506 or rapamycin, inhibitors of FKBP12 isomerase activity, reverse these stabilizing effects. These results provide the first natural cellular function for FKBP12, and establish that the functional Ca2+ release channel complex includes FKBP12.
The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiL, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 microns in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.
During maturation of hamster oocytes, the distribution of the endoplasmic reticulum (ER) and inositol 1,4,5-trisphosphate receptors (InsP3Rs) was found to change dramatically, as observed using confocal microscopy with DiI and electron microscopy for the ER and immunohistochemistry for InsP3Rs. In immature oocytes at the germinal vesicle (GV) stage, ER and InsP3Rs were located predominantly in several large masses near the surface and also in the perinuclear region near the surface. In contrast, fine ER networks and low-density InsP3Rs were present in the inner cytoplasm. The ER appeared to be formed as vesicles from annulate lamellae (AL) in the subcortical area. Rises in Ca2+ concentration occurred in the cytoplasm and the GV when immature oocytes were inseminated, but clear Ca2+ waves did not occur. Ca2+ rises began preferentially from the perinuclear region in response to low doses of serotonin or to uniform stimulation of InsP3Rs with photocleavage of caged InsP3. Serum also induced inhomogeneous Ca2+ release, shown by nonpropagating Ca2+ rises at multiple surface sites. Between the GV stage and prometaphase I the number and size of the surface ER masses increased, and the AL disappeared. This quantitative ER maturation was followed by a second step, spatial maturation. After prometaphase I, surface ER masses gradually dispersed to a number of much smaller ER clusters near the surface and, together with the perinuclear mass, were incorporated into thicker ER networks, resulting in a reticular pattern of the ER with small patches of InsP3Rs throughout the mature egg. The ER shifted to the peripheral surface with apposition to cortical granules. These developmental changes in ER Ca2+ stores may account, at least partly, for the acquisition of the ability of an egg to undergo normal fertilization.
1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis in Xenopus laevis oocytes and eggs in response to cytosolic Ca2+. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells. 2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca2+ induced by incubation with ionomycin. 3. Elevated Ca2+ triggered cortical granule exocytosis in eggs but not in oocytes. 4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules. 5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in both Xenopus laevis oocytes and X. laevis eggs (Bement, W. M., and Capco, D. G., J. Cell Biol. 108, 885-892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both. 6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca2+ stimulus. In the oocytes, cortical granule components necessary for Ca(2+)-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca2+ stimulus and downstream events.
The purpose of this study was to determine at what stage of meiotic maturation mouse oocytes develop the ability to undergo sperm-induced activation, assayed by cortical granule (CG) release. Germinal vesicle breakdown (GVBD), prometaphase I (proMI), metaphase I (MI), and metaphase II (MII, the normal stage at fertilization) stages were evaluated by quantitative image analysis. At 2 hr, fertilized MII eggs underwent a mean CG loss of 69%; loss was global--in the entire cortex occupied by CGs. In contrast, fertilized GVBD and proMI oocytes had no significant CG loss in the cortex after 2 hr. After 4 hr, 63, 67, and 88% of fertilized GVBD, proMI, and MI oocytes, respectively, had localized CG release only in the vicinity of the fertilizing sperm. At 4 hr, GVBD oocytes had small sperm-associated, CG-depleted domains, approximately 100 microns 2 (< 1% of the oocyte cortex), whereas those for proMI oocytes were approximately 2500 microns 2. The CG density was reduced by 90% in these domains, whereas the remaining cortex showed no evidence of CG loss. Both the extent and time course of CG loss were altered in oocytes fertilized before MII and were not significantly affected with 25 microM thimerosal, which sensitizes egg calcium stores. Unlike the ability to undergo CG release, the ability to initiate sperm chromatin decondensation was not stage specific and the extent of decondensation was cell cycle related. Competence to undergo CG loss develops in two phases: the ability to undergo localized CG release increases between the GV and MI stages, whereas the mechanism of propagating a normal wave of global CG loss from the site of sperm entry develops between MI and MII.
Inositol 1,4,5-trisphosphate receptors (InsP3R) in Xenopus laevis oocytes were localized and their regulation by Ca2+ was investigated. Antibodies raised against the C-terminal region of the mouse cerebellar InsP3R (cAb) cross-reacted with a 255 kD protein in Western blots of Xenopus microsomal membranes. Immunolocalization of this protein in cryosections of oocytes revealed diffuse staining of the cytoplasm, intense staining of the sub-plasma membrane region of the animal hemisphere, and punctate staining in association with the germinal vesicle. In the presence of 40 microM free Ca2+, isolated oocyte membranes exhibited a high affinity binding site for Ins 1,4,5-P3 (KD = 5nM) and a binding capacity of 450 fmol/mg protein. The specific binding capacity of oocyte membranes for [3H]-Ins 1,4,5-P3 increased as the level of free Ca2+ present in binding assays was raised from < 0.1 nM to 4.0 microM, with an apparent EC50 of 60 nM. Increasing the concentration of free Ba2+ failed to facilitate [3H]-Ins1,4,5-P3 binding. Other inositol phosphates competed for Ins1,4,5-P3 binding sites with approximate IC50 values of: Ins1,3,4,5-P4 = 79 nM, Ins2,4,5-P3 = 455 nM and L-Ins1,4,5-P3 = 20 microM. In addition, 150 micrograms/ml (approximately 12 microM) heparin displaced 50% of bound [3H]-Ins1,4,5-P3, whereas caffeine (10 mM) had little effect. Functional reconstitution of solubilized InsP3Rs into lipid bilayers revealed that Ca2+ was a necessary co-agonist for activation of the InsP3R. When InsP3 (5 microM) and Ca2+ (5 microM) were applied together, conductance steps were observed. InsP3 or Ca2+ alone had little effect. These results suggest that the subcellular organization of InsP3Rs and the facilitation of InsP3 binding and channel opening by Ca2+ contribute to the Ins1,4,5-P3-mediated Ca2+ spikes, waves, and oscillations observed in Xenopus oocytes.
Although the important subplasmalemmal localization of cortical granules (CG) has been documented, little is known about the timing and mechanism of granule translocation to the oocyte cortex. A fluorescent CG probe, confocal microscopy, and image analysis were used to obtain kinetic, spatial, and temporal information about CG migration during mouse oogenesis. The mean CG density in the cortex increased exponentially, from 3 to 14 CG/100 microns 2, during oocyte growth from 40-50 microns to 70-80 microns in diameter, respectively. Full-grown, > or = 80-microns, germinal vesicle-intact oocytes had a density of 30-35 CG/100 microns 2. During growth from 40-50 microns to > or = 80 microns, the mean total number of CG in the cortex per oocyte increased from < 500 to > 6000. When analyzed in terms of the stage of germinal vesicle chromatin organization, the mean CG density increased from 3 to 21 CG/100 microns 2 from stage I to early stage IV, respectively. In 50-60-microns oocytes, there was a crescent-shaped area of perinuclear staining containing both granules and Golgi apparatus-like structures, which were also more sparsely distributed in the subcortical cytoplasm. It is likely that these were sites of CG production from which translocation takes place; they were not observed in ovulated metaphase II eggs. This study demonstrates that CG number in the cortex increases continually during mouse oocyte growth (in contrast to that in several other species), that there is a greater than twofold increase in CG number during the final phase of growth, and that subcortical production ceases after ovulation.
A study was carried out to determine whether pig cortical granules (CGs) could be visualized using fluorescein isothiocyanate (FITC)-labelled lectins. Following labelling with FITC-labelled peanut agglutinin (FITC-PNA), fluorescent spots were observed that had a distribution during maturation and fertilization entirely consistent with that observed by electron microscopy. For the first 18 h of in vitro maturation, most of the fluorescent spots of FITC-PNA were distributed throughout the cortical cytoplasm. Thereafter, the CGs underwent centrifugal migration to form a monolayer next to the plasma membrane. Following penetration by sperm, fluorescent spots were extruded into the perivitelline space, where they aggregated forming fluorescent clumps, which subsequently formed a reticulate structure surrounding the egg. Fluorescence was gradually lost such that by 18 h after insemination none could be detected in 70% of the eggs. The results indicate that CGs in pig oocytes contain galactosyl-rich glycoconjugates and that FITC-PNA is a useful probe for their rapid visualization and examination.
Fertilization of the mammalian egg initiates transient and repetitive release of Ca2+ from intracellular stores. The mechanism by which these Ca2+ transients are produced is not completely known. We examined the role of two principal Ca2+ release mechanisms, inositol trisphosphate-induced Ca2+ release and Ca(2+)-induced Ca2+ release, in altering intracellular Ca2+ in the mouse egg. Microinjection of inositol 1,4,5-trisphosphate (IP3) transiently elevated intracellular Ca2+ and, at higher concentrations, produced repetitive Ca2+ transients. Addition of 100 microM thimerosal, a sulfhydryl reagent, caused repetitive Ca2+ transients. IP3 and thimerosal responses were inhibited by prior injection of heparin, a competitive antagonist of IP3-induced Ca2+ release. Addition of caffeine or injection of caffeine, ryanodine, or cyclic ADP-ribose, which are known to initiate or modulate Ca(2+)-induced Ca2+ release in sea urchin eggs and other cells, produced no change in intracellular Ca2+. The response to injection of Ca2+ was not altered by prior injection of ryanodine. The magnitude of the Ca2+ transients produced by injection of IP3 was not changed by prior injection of cyclic ADP-ribose or external caffeine. We found no evidence of Ca(2+)-induced Ca2+ release from ryanodine- or caffeine-sensitive stores. It is most likely that release of Ca2+ from intracellular stores in the mouse egg is dependent on IP3-induced Ca2+ release.
The presence of inositol 1,4,5-triphosphate receptor (InsP3R), calreticulin, and calsequestrin was demonstrated in eggs of sea urchins (Lytechinus pictus, Lytechinus variegatus, and Strongylocentroutus purpuratus) and Xenopus laevis. Binding of inositol 1,4,5-trisphosphate (InsP3) to microsomes of L. pictus eggs was inhibited by heparin and NaCl. An affinity-purified antibody against the C-terminal of the type I InsP3R, which recognizes InsP3R isoforms of rabbit brain (273 kDa) and Xenopus oocytes and eggs (256 kDa), reacted with a 373-kDa protein in sea urchin eggs. The 373-kDa protein was tentatively identified as the sea urchin egg InsP3R. Observations with fluorescence microscopy indicated that the InsP3R is present throughout the cytoplasm of sea urchin eggs in a pattern consistent with the distribution of endoplasmic reticulum. Small differences in the relative amount of reaction deposits in cortex vs subcortex were noted among the species of sea urchins examined. Reaction product was also localized to the periphery of female pronuclei in eggs of all three sea urchins. InsP3R reactivity was present in the perinuclear region, along the periphery of the germinal vesicle, and throughout the animal and vegetal hemispheres of Xenopus oocytes. A similar cytoplasmic staining pattern was also observed in eggs, although islands of reactivity, much larger than those in oocytes, were present in the animal hemisphere of eggs. Calreticulin and calsequestrin in sea urchin eggs had the same molecular mass as in rabbit brain (56 and 60 kDa, respectively), but differed from those present in Xenopus oocytes/eggs (61 and 57 kDa, respectively). The distribution of calreticulin and calsequestrin in both sea urchin and Xenopus oocytes and eggs was similar to that observed for the InsP3R. These results are discussed in relation to previous studies of Ca2+ regulation during egg development and fertilization and suggest that in the oocytes and eggs of the species examined, InsP3-sensitive Ca2+ stores play an important role in the regulation of cellular Ca2+.
To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed polarized distribution of XIP3R in the cytoplasm of the animal hemisphere in a well-organized endoplasmic reticulum-like structure and intensive localization in the perinuclear region of stage VI immature oocytes. In ovulated unfertilized eggs, XIP3R was densely enriched in the cortical region of both hemispheres in addition to its polarized localization. After fertilization, XIP3R showed a drastic change in its distribution in the cortical region. These results imply the predominant role of the XIP3R in both the formation and propagation of Ca2+ waves at fertilization.