BAPTA‐AM dramatically improves maturation and development of bovine oocytes from grade‐3 cumulus‐oocyte complexes

Intracellular free calcium ([Ca²⁺]_i) is essential for oocyte maturation and early embryonic development. Here, we investigated the role of [Ca²⁺]_i in oocytes from cumulus‐oocyte complexes (COCs) with respect to maturation and early embryonic development, using the calcium‐buffering agent BAPTA‐AM (1,2‐bis[2‐aminophenoxy]ethane‐N,N,N′,N′‐tetraacetic acid tetrakis [acetoxymethyl ester]). COCs were graded based on compactness of the cumulus mass and appearance of the cytoplasm, with Grade 1 indicating higher quality and developmental potential than Grade 3. Results showed that: (i) [Ca²⁺]_i in metaphase‐II (MII) oocytes from Grade‐3 COCs was significantly higher than those from Grade‐1 COCs, and was significantly reduced by BAPTA‐AM; (ii) nuclear maturation of oocytes from Grade‐3 COCs treated with BAPTA‐AM was enhanced compared to untreated COCs; (iii) protein abundance of Cyclin B and oocyte‐specific Histone 1 (H1FOO) was improved in MII oocytes from Grade‐3 COCs treated with BAPTA‐AM; (iv) Ca²⁺ transients were triggered in each group upon fertilization, and the amplitude of [Ca²⁺]_i oscillations increased in the Grade‐3 group upon treatment with BAPTA‐AM, with the magnitude approaching that of the Grade‐1 group; and (v) cleavage rates and blastocyst‐formation rates were improved in the Grade‐3 group treated with BAPTA‐AM compared to untreated controls following in vitro fertilization and parthenogenetic activation. Therefore, BAPTA‐AM dramatically improved oocyte maturation, oocyte quality, and embryonic development of oocytes from Grade‐3 COCs.     

Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca2+ channel, is a novel substrate of polo-like kinase 1 in eggs

To initiate embryo development, the sperm induces in the egg release of intracellular calcium ([Ca²⁺]_i). During oocyte maturation, the inositol 1,4,5-trisphosphate receptor (IP₃R1), the channel implicated, undergoes modifications that enhance its function. We found that IP₃R1 becomes phosphorylated during maturation at an MPM-2 epitope and that this persists until the fertilization-associated [Ca²⁺]_i responses cease. We also reported that maturation without ERK activity diminishes IP₃R1 MPM-2 reactivity and [Ca²⁺]_i responses. Here, we show that IP₃R1 is a novel target for Polo-like kinase1 (Plk1), a conserved M-phase kinase, which phosphorylates it at an MPM-2 epitope. Plk1 and IP₃R1 interact in an M-phase preferential manner, and they exhibit close co-localization in the spindle/spindle poles area. This co-localization is reduced in the absence of ERK activity, as the ERK pathway regulates spindle organization and IP₃R1 cortical re-distribution. We propose that IP₃R1 phosphorylation by Plk1, and possibly by other M-phase kinases, underlies the delivery of spatially and temporally regulated [Ca²⁺]_i signals during meiosis/mitosis and cytokinesis.     

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP₃) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmannet al.,1995,Dev. Biol.170, 607–615). Here, we investigated whether there is an increase in the number of available IP₃receptors after maturation and whether there is a redistribution of IP₃receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP₃receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP₃receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP₃-induced Ca²⁺release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca²⁺release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP₃, suggesting that eggs contain a greater number of functional IP₃receptors. Immunolocalization of the IP₃receptor revealed that receptors were present in large clusters, 1–2 μm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 μm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP₃receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca²⁺release at fertilization.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Changes of intracellular free calcium during intracytoplasmic sperm injection

The present experiments were undertaken to investigate whether the procedure of intracytoplasmic sperm injection (ICSI) is associated with changes in the intracellular free calcium concentration ([Ca²⁺]_i). [Ca²⁺]_i was measured, using the calcium-sensitive dye fura-2, during and after impalement of mouse oocytes with an ICSI pipette and injection of a small amount of medium alone or of medium containing a normal human spermatozoon. Forty-five oocytes were injected with medium. Two different responses were observed: 20 of these cells showed a large increase of [Ca²⁺]_i upon impalement; the other 25 cells did not show any change of [Ca²⁺]_i, neither in the acute period nor in a late period 4 hr after impalement. All the cells that responded with an increase of [Ca²⁺]_i subsequently lysed within the first 30 min following impalement, while all the cells with no [Ca²⁺]_i change remained intact. This observation suggests that only traumatic impalement is associated with an increase of [Ca²⁺]_i. Thirty-one oocytes were successfully, i.e., without subsequent cell lysis, injected with a normal mouse or human spermatozoon. In none of these cells could any acute or late change of [Ca²⁺]_i be observed. The experiments illustrate that successful performance of the ICSI procedure, i.e., ICSI not followed by cell lysis, is not associated with changes of [Ca²⁺]_i in mouse oocytes. This suggests that the ICSI technique, by itself, does not help in activating the oocyte via manipulation-induced changes of [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca2+ influx

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca²⁺) concentration ([Ca²⁺]_i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca²⁺]_i via blocking the influx of extracellular Ca²⁺ or chelating ooplasmic free Ca²⁺. After IVM, cumulus–oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca²⁺]_i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus–oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca²⁺]_i was significantly decreased. When oocytes were cultured in Ca²⁺-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca²⁺]_i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca²⁺]_i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca²⁺ or reducing ooplasmic free Ca²⁺. 1-Octanol, BAPTA-AM, or Ca²⁺-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.     

Evaluation of developmental competence,nuclear and ooplasmic maturation of calf oocytes

In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca²⁺ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca²⁺ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP₃), used to test the sensitivity of the InsP₃R, released significantly less Ca²⁺ in calf than in cow oocytes, whereas higher concentrations of InsP₃ (500 μM) released maximal [Ca²⁺]i in both oocytes. These results suggested that the Ca²⁺ content of intracellular stores was similar, but the sensitivity of the InsP₃R may be different. Following insemination, calf oocytes exhibiting [Ca²⁺]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation. © 1996 Wiley-Liss, Inc.     

Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation

Egg activation and further embryo development require a sperm-induced intracellular Ca²⁺ signal at the time of fertilization. Prior to fertilization, the egg's Ca²⁺ machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca²⁺ releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1), which is responsible for most of this Ca²⁺ release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP₃R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP₃R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T²⁶⁵⁶ as a major Plk1 site on IP₃R1. We therefore propose that the initial increase in IP₃R1 sensitivity during oocyte maturation is underpinned by IP₃R1 phosphorylation at an MPM-2 epitope(s).     

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

Aging-related changes in calcium oscillations in fertilized mouse oocytes

Aging of oocytes, being not fertilized after ovulation for a prolonged time, considerably affects normal development of the fertilized oocyte. We examined effects of the aging on a series of highly repetitive Ca²⁺ transients commonly seen in fertilized mouse oocytes (Ca²⁺ oscillations). Frequency of Ca²⁺ oscillations in the aged oocyte [20 hrs after induction of superovulation by i.p. human chorionic gonadotropin (hCG)] was significantly higher (34.1 ± 5.8 1/hr) than the fresh oocyte (14 hr post-hCG, 21.8 ± 7.9 1/hr). Rates of rise and fall of individual Ca²⁺ transient in the aged oocyte were significantly slower than the fresh oocyte, whereas durations of individual Ca²⁺ transients were similar. When extracellular Ca²⁺ was raised from 2.04 mM to 5.00 mM, aged oocytes showed significant prolongation of the duration of individual Ca²⁺ transient, that resulted in a sustained elevation of intracellular Ca²⁺ ([Ca²⁺]_i) in 33% of the aged oocyte. Transient increase in [Ca²⁺]_i by photolysis of a caged Ca²⁺, Nitr-5, injected into cytoplasm was completely restored in the fresh oocyte [fluorescence intensity of [Ca²⁺]_i indicator dye Fluo-3 (F₄₈₀) returned to 97 ± 2% of the control level, time constant = 37 ± 9 sec]. In contrast, in the aged oocyte, restoration of F₄₈₀ following Nitr-5 photolysis was incomplete (115 ± 12% of the control) and slow (time constant = 64 ± 23 sec). Because inhibition of the Ca²⁺ pump of the endoplasmic reticulum (ER) by 5 μM thapsigargin almost completely inhibited restoration of F₄₈₀ following Nitr-5 photolysis in the fresh oocyte, we conclude that the aging-related changes in Ca²⁺ oscillations may be accounted for by dysfunction of intracellular Ca²⁺ regulation, presumably of the Ca²⁺ pump of the ER. Mol. Reprod. Dev. 48:383–390, 1997. © 1997 Wiley-Liss, Inc.     

Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca²⁺ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca²⁺]_i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP₃R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP₃. At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca²⁺]_i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min⁻¹). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP₃R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP₃R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca²⁺ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca²⁺ signaling, each of them generating oscillations with a specific temporal pattern.     

Ca<Superscript>2+</Superscript> regulation in the absence of the <Emphasis Type="Italic">iplA</Emphasis> gene product in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The [Ca²⁺]_i-change is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-entry. The significance of the [Ca²⁺]_i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca²⁺-buffers in the cytosol indicates that a [Ca²⁺]_i-increase is required for chemotaxis. Yet, the iplA ^- mutant disrupted in a gene bearing similarity to IP₃-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca²⁺]_i-transient which favours the view that [Ca²⁺]_i-changes are insignificant for chemotaxis.     

Kinase-dependent Regulation of Inositol 1,4,5-Trisphosphate-dependent Ca2+ Release during Oocyte Maturation

Fertilization induces a species-specific Ca²⁺ transient with specialized spatial and temporal dynamics, which are essential to temporally encode egg activation events such as the block to polyspermy and resumption of meiosis. Eggs acquire the competence to produce the fertilization-specific Ca²⁺ transient during oocyte maturation, which encompasses dramatic potentiation of inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release. Here we show that increased IP₃ receptor (IP₃R) sensitivity is initiated at the germinal vesicle breakdown stage of maturation, which correlates with maturation promoting factor (MPF) activation. Extensive phosphopeptide mapping of the IP₃R resulted in ∼70% coverage and identified three residues, Thr-931, Thr-1136, and Ser-114, which are specifically phos pho ryl a ted during maturation. Phospho-specific antibody analyses show that Thr-1136 phos pho ryl a tion requires MPF activation. Activation of either MPF or the mitogen-activated protein kinase cascade independently, functionally sensitizes IP₃-dependent Ca²⁺ release. Collectively, these data argue that the kinase cascades driving meiotic maturation potentiates IP₃-dependent Ca²⁺ release, possibly trough direct phos pho ryl a tion of the IP₃R.Egg activation refers to the cellular and molecular events that take place immediately following fertilization, transitioning the zygote into embryogenesis. In vertebrates, egg activation encompasses the block to polyspermy and the completion of oocyte meiosis, which is coupled to the extrusion of the second polar body. Interestingly, in all sexually reproducing organisms tested to date the cellular events associated with egg activation are Ca²⁺-dependent (1). Importantly the Ca²⁺ signal at fertilization encodes the progression of these cellular events in a defined temporal sequence that ensures a functional egg-to-embryo transition (2, 3). The first order of business for the fertilized egg is to block polyspermy, which could be lethal to the embryo. This presents a particularly difficult problem for the large Xenopus oocyte. Therefore, this species employs a fast and slow blocks to polyspermy, both of which are Ca²⁺-dependent (4). In addition, the Ca²⁺ release wave at fertilization releases the metaphase II cytostatic factor-dependent arrest in Xenopus oocytes. As is the case in other vertebrates, Xenopus eggs arrest at metaphase of meiosis II, an event that marks the completion of maturation.Therefore, Ca²⁺ dynamics at fertilization initiate and temporally encode critical cellular events for the egg-to-embryo transition. Specificity in Ca²⁺ signaling is encoded to a large extent in the spatial, temporal, and amplitude features of the Ca²⁺ signal. This endows Ca²⁺ signaling with its versatility and specificity, where in the same cell Ca²⁺ signals can mediate distinct cellular responses (5, 6).Ca²⁺ signaling pathways and intracellular organelles remodel during oocyte maturation, a complex cellular differentiation that prepares the egg for fertilization and egg activation (7, 8). In Xenopus the activity and distribution of multiple essential Ca²⁺-transporting proteins is modulated dramatically during oocyte maturation (8). Functional studies and mathematical modeling support the conclusion that the two critical determinants of Ca²⁺ signaling remodeling during Xenopus oocyte maturation are the internalization of the plasma-membrane Ca²⁺-ATPase, and the sensitization of inositol 1,4,5-trisphosphate (IP₃)²-dependent Ca²⁺ release (9–11). Indeed Ca²⁺ release from intracellular stores through the IP₃ receptor (IP₃R) represents the primary source for the initial Ca²⁺ rise at fertilization in vertebrates (12–14). The sensitivity of IP₃-dependent Ca²⁺ release is enhanced during maturation (10, 15). The IP₃R physically clusters during maturation (9, 16), and this is associated with functional clustering of elementary Ca²⁺ release events (10). IP₃R clustering is important for the slow and continuous nature of Ca²⁺ wave propagation in Xenopus eggs (10). In fact the potentiation of IP₃-dependent Ca²⁺ release is a hallmark of Ca²⁺ signaling differentiation during oocyte maturation in several vertebrate and invertebrate species (17–19). However, the mechanisms underlying enhanced IP₃-dependent Ca²⁺ release are not well understood.An attractive mechanism to explain increased IP₃R sensitivity during oocyte maturation is phosphorylation, given the critical role kinase cascades play in the initiation and progression of the meiotic cell cycle. Furthermore, the affinity of the IP₃R increases during mitosis apparently due to direct phosphorylation by maturation-promoting factor (MPF) (20, 21). In contrast, in starfish eggs, although the increase in Ca²⁺ release was dependent on MPF activation, MPF does not directly phosphorylate the IP₃R, but rather it appears to mediate its effect through the actin cytoskeleton (22, 23). More recently, the MAPK cascade has been shown to be important for shaping Ca²⁺ dynamics in mouse eggs (24). Together, these results argue that phosphorylation plays an important role in the sensitization of IP₃-dependent Ca²⁺ release during M-phase.Xenopus oocyte maturation is initiated by steroids that appear to act on a cell surface receptor (25). An important kinase cascade activated during maturation is the MAPK cascade that is initiated through the accumulation of Mos (Fig. 1A). This cascade culminates in the inhibition of Myt1, which phosphorylates and inhibits MPF. MPF is the key regulator of entry into M-phase and is composed of a Ser/Thr kinase subunit (cdk1) and cyclin B as a regulatory subunit. In addition, activation of Cdc25C is essential for oocyte maturation, because it represents the rate-limiting step in MPF activation (26). Cdc25C is phosphorylated by polo-like kinase through unknown upstream steps. In this work we analyze the functional regulation and phosphorylation pattern of the IP₃R during oocyte maturation to better understand the role of cell cycle kinases in modulating IP₃-dependent Ca²⁺ release.Open in a separate windowFIGURE 1.IP₃-dependent Ca²⁺ release dynamics during maturation. A, kinase cascades driving Xenopus oocyte maturation. B, oocytes were injected with caged-IP₃ and Oregan Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis 1 before imaging. Maturation was induced with progesterone, and cells were collected at different time points as indicated. Cells were imaged in line scan mode on a Zeiss LSM510 with the near UV 450 nm laser continuously on, at low intensity to produce a slow gradual IP₃ rise. After imaging each cell was lysed and analyzed individually for the activation state of MAPK and MPF. MPF was assayed using an anti-phospho-Tyr-15-cdk1 antibody (arrow). Dephosphorylation is indicative of MPF activation. MAPK activation was detected using a phospho-specific MAPK antibody (arrowhead). Tubulin was the loading control (dash). C, percent of cells at each time point that either exhibit no release for the duration of the line scan (No Rel., black), puffs only (puffs, green), puffs followed by a wave (Puff-Wave, blue), or only a Ca²⁺ wave (Wave, red). For each time point n = 11–23 cells. D, amplitude of the first peak during the line scan as compared with the maximal Ca²⁺ signal. Mean ± S.E. (n = 9–18). E, latency until the first Ca²⁺ signal (Time to first peak) as compared with the time required to reach maximal signal (Time to Max). Mean ± S.E. (n = 9–18). For C–E: oocytes (Ooc); cells treated with progesterone that have not undergone GVBD at 2 or more hours after progesterone (p > 2); cells at GVBD and up to 0.5 h after GVBD (GVBD 0–0.5); cells from 0.5 to 2.5 h after GVBD (GVBD 0.5–2.5); fully mature eggs at 3 or more hours after GVBD (>3 egg).     

Mitochondrial regulation of [Ca2+]i oscillations during cell cycle resumption of the second meiosis of oocyte

Oocyte is arrested at metaphase of the second meiosis until fertilization switching on [Ca²⁺]i oscillations. Oocyte activation inefficiency is the most challenging problem for failed fertilization and embryonic development. Mitochondrial function and intracellular [Ca²⁺]i oscillations are two critical factors for the oocyte’s developmental potential. We aimed to understand the possible correlation between mitochondrial function and [Ca²⁺]i oscillations in oocytes. To this end, mitochondrial uncoupler CCCP which damages mitochondrial function and two small molecule mitochondrial agonists, L-carnitine (LC) and BGP-15, were used to examine the regulation of [Ca²⁺]i by mitochondrial functions. With increasing CCCP concentrations, [Ca²⁺]i oscillations were gradually diminished and high concentrations of CCCP led to oocyte death. LC enhanced mitochondrial membrane potential and [Ca²⁺]i oscillations and even improved the damage induced by CCCP, however, BGP-15 had no beneficial effect on oocyte activation. We have found that mitochondrial function plays a vital role in the generation of [Ca²⁺]i oscillations in oocytes, and thus mitochondria may interact with the ER to generate [Ca²⁺]i oscillations during oocyte activation. Improvement of mitochondrial functions with small molecules can be expected to improve oocyte activation and embryonic development in infertile patients without invasive micromanipulation.     

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A_2A receptors (A_2A R) coupled to G_s/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A_2A R in the modulation of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) involving IP₃, cAMP and PKA was investigated in HEK 293-A_2A R. The levels of IP₃ and cAMP were observed by enzyme immunoassay detection method and [Ca²⁺]_i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A_2A R agonist NECA showed increased levels of cAMP, PKA, IP₃ and [Ca²⁺]_i levels. However, the reverse effect was observed with A_2A R antagonists (ZM241385 and caffeine). Blocking the G_αq/PLC/DAG/IP₃ pathway with neomycin, a PLC inhibitor did not affect the modulation of IP₃ and [Ca²⁺]_i levels in HEK 293-A_2A R cells. To investigate the G_αi/AC/cAMP/PKA, HEK 293-A_2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A_2A R agonist (NECA) showed no effect on cAMP levels. Further, G_αs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP₃ mediated [Ca²⁺]_i modulation. In the HEK 293-A_2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP₃ and [Ca²⁺]_i levels. The study distinctly demonstrated that A_2A R modulates IP₃ levels to release the [Ca²⁺]_i via cAMP-dependent PKA. The role of A_2A R mediated G_αs pathway inducing IP₃ mediated [Ca²⁺]_i release may open new avenues in the therapy of neurodegenerative disorder.     

GT-repeat polymorphism in the heme oxygenase-1 gene promoter and the risk of carotid atherosclerosis related to arsenic exposure

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Endoplasmic Reticulum Dysfunction and Ca<Superscript>2<Emphasis Type="Bold">+</Emphasis></Superscript> Deregulation in Isolated CA1 Neurons during Oxygen and Glucose Deprivation

Intracellular calcium ([Ca²⁺]_i) plays a pivotal role in neuronal ischemia. The aim of the present study was to investigate the routes of Ca²⁺ entry during non-excitotoxic oxygen and glucose deprivation (OGD) in acutely dissociated rat CA1 neurons. During OGD the fluo-3/fura red ratio reflecting [Ca²⁺]_i increased rapidly and irreversibly. [Ca²⁺]_i increased to the same degree in Ca²⁺ depleted medium, and also when both the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate (IP₃) receptors were blocked. When the endoplasmic reticulum (ER) Ca²⁺ stores were emptied with thapsigargin no increase in [Ca²⁺]_i was observed independent of extracellular Ca²⁺. The OGD induced Ca²⁺ deregulation in isolated CA1 neurons is not prevented by removing Ca²⁺, or by blocking the IP₃– or RyR receptors. However, when SERCA was blocked, no increase in [Ca²⁺]_i was observed suggesting that SERCA dysfunction represents an important mechanism for ischemic Ca²⁺ overload.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.