Aged mouse oocytes fail to readjust intracellular adenosine triphosphates at fertilization

Postovulatory aging of oocytes significantly affects embryonic development. Also, altered Ca2+ oscillation patterns can be observed in fertilized, aged mouse oocytes. Because Ca2+ oscillations depend on Ca2+ release and reuptake in the endoplasmic reticulum, and the latter relies on ATP availability, we simultaneously measured changes in intracellular ATP concentration ([ATP]i) and Ca2+ oscillations in fresh and aged mouse oocytes. We continuously assessed changes in [ATP]i from intracellular free Mg2+ concentration measured by fluorescent dye Magnesium Green (MgG) while intracellular Ca2+ concentration ([Ca2+]i) was monitored by Fura-PE3. At fertilization, MgG fluorescence was transiently increased concomitant with the first transient elevation in [Ca2+]i, indicating a relative decrease in [ATP]i. In fresh oocytes, it was quickly followed by a significant decrease below baseline, indicating a relative increase in [ATP]i. In contrast, in aged oocytes, such a decrease in MgG fluorescence was not observed. In a separate experiment, ATP content in fresh and aged oocytes was determined in vitro by the luciferin-luciferase assay. Intracellular ATP contents measured in vitro were comparable in unfertilized fresh and aged oocytes. Intracellular ATP content at 5 h after fertilization was increased in both oocytes, where fresh oocytes showed a significantly higher intracellular value than aged oocytes. These findings suggest that aged mouse oocytes fail to readjust the level of intracellular ATP at fertilization. Relative deficiencies of ATP at fertilization might lead to an altered Ca2+ oscillation pattern and poor developmental potency, which is commonly noted in aged oocytes.     

Effects of aging on inositol 1,4,5-triphosphate-induced Ca(2+) release in unfertilized mouse oocytes

We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca(2+)-ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383-390). We undertook the present study to further examine the effects of oocyte aging on Ca(2+) release from the inositol 1,4,5-triphosphate (InsP(3))-sensitive Ca(2+) channels of the ER membrane, because not only Ca(2+) reuptake, but also Ca(2+) release from the ER, substantially affect Ca(2+) oscillations in fertilized oocytes. A transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was induced by photolysis of caged InsP(3) microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca(2+)](i) significantly decreased in the aged oocytes. Reduced ER Ca(2+) release in the aged oocyte may not be attributable to aging-related desensitization of the InsP(3)-sensitive Ca(2+) channels in the ER because concentrations of caged InsP(3) for half maximal [Ca(2+)](i) increase were identical for fresh and aged oocytes. The peak [Ca(2+)](i) response following administration of 5 microM thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca(2+) stores. We conclude from these results that reduction of Ca(2+) release from the InsP(3)-sensitive Ca(2+) stores in the aged oocyte arises from depletion of the ER Ca(2+) stores with aging. These aging-related changes in Ca(2+) release and reuptake may account for alterations in Ca(2+) oscillations in aged fertilized oocytes.     

Animal-vegetal axis patterning mechanisms in the early sea urchin embryo

During mouse fertilization the spermatozoon induces a series of low-frequency long-lasting Ca(2+) oscillations. It is generally accepted that these oscillations are due to Ca(2+) release through the inositol 1,4,5-trisphosphate (InsP(3)) receptor. However, InsP(3) microinjection does not mimic sperm-induced Ca(2+) oscillations, leading to the suggestion that the spermatozoon causes Ca(2+) release by sensitizing the InsP(3) receptor to basal levels of InsP(3). This contradicts recent evidence that the spermatozoon triggers Ca(2+) oscillations by introducing a phospholipase C or else an activator of phospholipase C. Here we show for the first time that sperm-induced Ca(2+) oscillations may be mimicked by the photolysis of caged InsP(3) in both mouse metaphase II eggs and germinal vesicle stage oocytes. Eggs, and also oocytes that had displayed spontaneous Ca(2+) oscillations, gave long-lasting Ca(2+) oscillations when fertilized or when caged InsP(3) was photolyzed. In contrast, oocytes that had shown no spontaneous Ca(2+) oscillations did not generate many oscillations when fertilized or following photolysis of caged InsP(3). Fertilization in eggs was most closely mimicked when InsP(3) was uncaged at relatively low amounts for extended periods. Here we observed an initial Ca(2+) transient with superimposed spikes, followed by a series of single transients with a low frequency; all characteristics of the Ca(2+) changes at fertilization. We therefore show that InsP(3) can mimic the distinctive pattern of Ca(2+) release in mammalian eggs at fertilization. It is proposed that a sperm Ca(2+)-releasing factor operates by generating a continuous small amount of InsP(3) over an extended period of time, consistent with the evidence for the involvement of a phospholipase C.     

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.     

Ability of integrins to mediate fertilization, intracellular calcium release, and parthenogenetic development in bovine oocytes

The ability of arginine-glycine-aspartic acid (RGD; a sequence recognized by integrins) or non-RGD-containing peptides to block fertilization, induce intracellular Ca(2+) oscillations, and initiate parthenogenetic development in bovine oocytes was investigated. Addition of a soluble RGD peptide during fertilization at concentrations ranging from 10 to 1000 microg/ml significantly decreased (P<0.05) fertilization as compared to the in vitro-fertilized controls. The addition of non-RGD peptide had no effect on fertilization. Two intracellular Ca(2+) transients 21.5+/- 1.9 min apart were observed in 56 of 60 oocytes incubated in RGD peptide concentrations ranging from 20 to 1000 microg/ml. No intracellular Ca(2+) transients were observed in medium alone, non-RGD treatment groups or in the RGD peptide at 10 microg/ml. The percentage of oocytes activated with ionomycin and 6-dimethylaminopurine (63% cleavage and 34% blastocyst development) was significantly higher (P<0.05) than those activated with the RGD peptide and 6-dimethylaminopurine (35% cleavage and 19% blastocyst development). These groups were significantly higher (P<0.05) than either peptide alone, 6-dimethylaminopurine alone, or the non-RGD peptide and 6-dimethylaminopurine treatment groups. These data provide evidence that ligation of an integrin on bovine oocytes with a soluble RGD peptide is capable of blocking fertilization, inducing intracellular Ca(2+) transients, and initiating parthenogenetic development.     

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei

In mammals, the sperm triggers a series of cytosolic Ca(2+) oscillations that continue for approximately 4 hours, stopping close to the time of pronucleus formation. Ca(2+) transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca(2+) signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca(2+) oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca(2+) oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca(2+) oscillations. A role for pronucleus formation in regulating Ca(2+) signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca(2+) oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin beta that inhibits nuclear transport, also prevents pronucleus formation and causes Ca(2+) oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca(2+) oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca(2+)-releasing activity contributes to the temporal organization of Ca(2+) transients in meiosis and mitosis in mice.     

The cortical endoplasmic reticulum (ER) of the mouse egg: localization of ER clusters in relation to the generation of repetitive calcium waves.

The endoplasmic reticulum (ER) of the mature mouse egg consists of a fine tubular network and pronounced accumulations in the cortex. The ER was visualized both in intact eggs and with in vitro preparations of the cortex using the fluorescent lipophilic dye, DiI. Immunofluorescent labeling of the ER in isolated cortical preparations demonstrated that the ER clusters contain inositol 1,4, 5-trisphosphate (IP(3)) receptors, indicating an important involvement in sperm-induced Ca(2+) transients, which are triggered by IP(3). We imaged the ER during fertilization and the subsequent Ca(2+) transients and found that the clusters remained intact throughout this period. Recovery of fluorescence after photobleaching established that the ER clusters are continuous with the reticular ER network and that these structures remain stable and continuous throughout the time of fertilization-induced Ca(2+) transients; continuity also remained during IP(3) injection. These results indicate that, in contrast to echinoderm eggs, the ER of mouse eggs does not become disrupted when it releases Ca(2+)at fertilization. The localization and apparent stability of the cortical ER clusters may be important in generating Ca(2+) oscillations, which are characteristic of fertilized mammalian eggs. Imaging of intracellular Ca(2+) revealed that Ca(2+) transients originate in the hemisphere of the egg that contains abundant ER clusters, thus the mouse contains a stable cortical pacemaker responsible for generating Ca(2+) waves.     

STIM1 is required for Ca2+ signaling during mammalian fertilization

During fertilization in mammals, a series of oscillations in the oocyte's intracellular free Ca(2+) concentration is responsible for oocyte activation and stimulation of embryonic development. The oscillations are associated with influx of Ca(2+) across the plasma membrane that is probably triggered by the depletion of the intracellular stores, a mechanism known as store-operated Ca(2+) entry. Recently, STIM1 has been identified in oocytes as a key component of the machinery that generates the Ca(2+) influx after store depletion. In this study, the involvement of STIM1 in the sperm-induced Ca(2+) oscillations and its significance in supporting subsequent embryo development were investigated. Downregulation of STIM1 levels in pig oocytes by siRNA completely inhibited the repetitive Ca(2+) signal triggered by the fertilizing sperm. In addition, a significantly lower percentage of oocytes cleaved or formed blastocysts when STIM1 was downregulated prior to fertilization compared to the control groups. Restoring STIM1 levels after fertilization in such oocytes by means of mRNA injection could not rescue embryonic development that in most cases was arrested at the 2-cell stage. On the other hand, STIM1 overexpression prior to fertilization did not alter the pattern of sperm-induced Ca(2+) oscillations and development of these fertilized oocytes up to the blastocyst stage was also similar to that registered in the control group. Finally, downregulation of STIM1 had no effect on oocyte activation when activation was stimulated artificially by inducing a single large elevation in the oocyte's intracellular free Ca(2+) concentration. These findings suggest that STIM1 is essential for normal fertilization as it is involved in the maintenance of the long-lasting repetitive Ca(2+) signal.     

Ca(2+) oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs

At fertilization in mammals, the sperm activates the egg by inducing a series of oscillations in the intracellular free Ca(2+) concentration. There is evidence showing that this oscillatory event is triggered by a sperm-derived protein factor which diffuses into egg cytoplasm after gamete membrane fusion. At present the identity of this factor and its precise mechanism of action is unknown. Here, we studied the specificity of action of the sperm factor in triggering Ca(2+) oscillations in mammalian eggs. In doing so, we examined the patterns of Ca(2+) signaling in mouse eggs, zygotes, parthenogenetic eggs and maturing oocytes following the stimulation of bovine sperm extracts which contain the sperm factor. It is observed that the sperm factor could induce Ca(2+) oscillations in metaphase eggs, maturing oocytes and parthenogenetically activated eggs but not in the zygotes. We present evidence that Ca(2+) oscillations induced by the sperm factor require a maternal machinery. This machinery functions only once in mammalian oocytes and eggs, and is inactivated by sperm-derived components but not by parthenogenetic activation. In addition, it is found that neither InsP(3) receptor sensitivity to InsP(3) nor Ca(2+) pool size are the determinants that cause the fertilized egg to lose its ability to generate sperm-factor-induced Ca(2+) oscillations at metaphase. In conclusion, our study suggests that the orderly sequence of Ca(2+) oscillations in mammalian eggs at fertilization is critically dependent upon the presence of a functional maternal machinery that determines whether the sperm-factor-induced Ca(2+) oscillations can persist.     

Maturation and fertilization in Lottia gigantea oocytes: intracellular pH, Ca(2+), and electrophysiology

Intracellular pH and Ca(2+) were measured with BCECF- and Calcium Green-dextran during maturation and fertilization of oocytes of the limpet Lottia gigantea. Maturation of oocytes from prophase to metaphase I of meiosis was induced in seawater adjusted to pH 9 with NH(4)OH. Intracellular pH rose during maturation induction, and maturation was also induced by microinjecting pH 8, but not pH 7, HEPES buffer. Intracellular Ca(2+) rose during NH(4)OH-induced maturation, but maturation was not inhibited when the increase was blocked by microinjection of BAPTA. When the metaphase I oocytes were fertilized(), there was an abrupt increase in intracellular Ca(2+), and activation (polar body formation) failed to occur in BAPTA-injected oocytes. Intracellular pH did not rise during fertilization. These observations show that maturation from prophase to metaphase I of meiosis is pH-dependent and activation of the metaphase I oocytes is Ca(2+)-dependent. A Ca(2+) action potential was present in both immature and mature oocytes but was more prominent in mature oocytes whose input resistance was higher. Fertilization produced a long-lasting (17-20 min) Na(+)-dependent fertilization potential with superimposed oscillations resembling Ca(2+) action potentials.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling.

BACKGROUND INFORMATION: The IP(3)R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca(2+) release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP(3)R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP(3)R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. RESULTS: In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP(3)R1 increased the rate of thapsigargin-mediated Ca(2+) leak and decreased the rate of Ca(2+) uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca(2+) stores. We detected the truncated IP(3)R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP(3)R1 blocked sperm factor-induced Ca(2+) oscillations and induced an apoptotic phenotype. CONCLUSIONS: In the present study, we show that caspase-3-mediated truncation of IP(3)R1 enhanced the Ca(2+) leak from the ER. We suggest a model in which, in normal conditions, the increased Ca(2+) leak is largely compensated by enhanced Ca(2+)-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca(2+) leak acts as a feed-forward mechanism to divert the cell into apoptosis.     

Calcium spark properties in ventricular myocytes are altered in aged mice

This study determined whether whole cell Ca(2+) transients and unitary sarcoplasmic reticulum (SR) Ca(2+) release events are constant throughout adult life or whether Ca(2+) release is altered in aging ventricular myocytes. Myocytes were isolated from young adult (approximately 5 mo old) and aged (approximately 24 mo old) mice. Spontaneous Ca(2+) sparks and Ca(2+) transients initiated by field stimulation were detected with fluo-4. All experiments were conducted at 37 degrees C. Ca(2+) transient amplitudes were reduced, and Ca(2+) transient rise times were abbreviated in aged cells stimulated at 8 Hz compared with young adult myocytes. Furthermore, the incidence and frequency of spontaneous Ca(2+) sparks were markedly higher in aged myocytes compared with young adult cells. Spark amplitudes and spatial widths were similar in young adult and aged myocytes. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Resting cytosolic Ca(2+) levels and SR Ca(2+) stores were assessed by rapid application of caffeine in fura-2-loaded cells. Neither resting Ca(2+) levels nor SR Ca(2+) content differed between young adult and aged cells. Thus increased spark frequency in aging cells was not attributable to increased SR Ca(2+) stores. Furthermore, the decrease in Ca(2+) transient amplitude was not due to a decrease in SR Ca(2+) load. These results demonstrate that alterations in fundamental SR Ca(2+) release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca(2+) release may reflect age-related changes in fundamental release events rather than changes in SR Ca(2+) stores and diastolic Ca(2+) levels.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.     

Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca(2+) or egg activation

Fertilization in mammals stimulates a series of Ca(2+) oscillations that continue for 3-4 h. Cell-cycle-dependent changes in the ability to release Ca(2+) are one mechanism that leads to the inhibition of Ca(2+) transients after fertilization. The downregulation of InsP(3)Rs at fertilization may be an additional mechanism for inhibiting Ca(2+) transients. In the present study we examine the mechanism of this InsP(3)R downregulation. We find that neither egg activation nor Ca(2+) transients are necessary or sufficient for the stimulation of InsP(3)R downregulation. First, parthenogenetic activation fails to stimulate downregulation. Second, downregulation persists when fertilization-induced Ca(2+) transients and egg activation are inhibited using BAPTA. Third, downregulation can be induced in immature oocytes that do not undergo egg activation. Other than fertilization, the only stimulus that downregulated InsP(3)Rs was microinjection of the potent InsP(3)R agonist adenophostin A. InsP(3)R downregulation was inhibited by the cysteine protease inhibitor ALLN but MG132 and lactacystin were not effective. Finally, we have injected maturing oocytes with adenophostin A and produced MII eggs depleted of InsP(3)Rs. We show that sperm-induced Ca(2+) signaling is inhibited in such InsP(3)R-depleted eggs. These data show that InsP(3)R binding is sufficient for downregulation and that Ca(2+) signaling at fertilization is mediated via the InsP(3)R.     

Oscillatory CaMKII activity in mouse egg activation

Fertilization-induced intracellular calcium (Ca(2+)) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these Ca(2+) signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in Ca(2+) similar to those at fertilization stimulate oscillatory Ca(2+)/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory Ca(2+) signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular Ca(2+) levels and CaMKII activity in the same population of eggs demonstrated a rapid and transient enzyme response to each increase in Ca(2+). Enzyme activity increased 370% during the first Ca(2+) rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time Ca(2+) reached its peak value. Single fertilized eggs monitored for Ca(2+) had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced Ca(2+) transients resulted in 39 and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of Ca(2+) and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of Ca(2+) in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation.     

Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

Fertilization and InsP3-induced Ca2+ release stimulate a persistent increase in the rate of degradation of cyclin B1 specifically in mature mouse oocytes

Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca2+. This increase in Ca2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca2+ can activate the destruction machinery during MI. We have investigated Ca2+ -induced cyclin destruction in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca2+. We find no evidence for a role for Ca2+ in MI since oocytes progress through MI in the absence of detectable Ca2+ transients. Furthermore, Ca2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. We conclude that increases in cytosolic Ca2+ induce transient changes in autofluorescence and that the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca2+ -induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca2+ acts to release the CSF-mediated brake on cyclin B1 destruction.     

Injection of sperm cytosolic factor into mouse metaphase II oocytes induces different developmental fates according to the frequency of [Ca(2+)](i) oscillations and oocyte age

Intracellular calcium ([Ca(2+)](i)) rises are a hallmark of mammalian fertilization and are associated with normal activation of embryonic development. Injection of mammalian sperm cytosolic factor (SCF) into oocytes has been shown to trigger [Ca(2+)](i) rises similar to those observed during fertilization, and to initiate normal embryonic development. However, Ca(2+) release has also been shown to be associated with cell death, but the mechanisms of the detrimental effects of Ca(2+) stimulation on development have not yet been investigated. Thus, studies were undertaken using SCF to test the effects of [Ca(2+)](i) oscillations on oocyte activation in freshly ovulated and aged oocytes. Injections of 1 mg/ml SCF into freshly ovulated mouse metaphase II oocytes, which evoked Ca(2+) responses with low frequency and short duration, induced normal activation and cleavage to the two-cell stage. Conversely, injection of 15 mg/ml SCF, which triggered high-frequency and persistent Ca(2+) responses, induced abnormal activation that was characterized by abnormal chromatin configurations, inhibition of DNA synthesis, and lack of first mitotic spindle assembly. More importantly, fertilization-like Ca(2+) responses induced by injection of 1 mg/ml SCF triggered cell death, rather than activation, in in vitro-aged oocytes. These oocytes exhibited extensive cytoplasmic and DNA fragmentation that was accompanied by activation of protein caspases, all of which are signs of apoptotic cell death. Fewer similarly aged oocytes that were either unstimulated or activated with 7% ethanol underwent fragmentation. Together, these results suggest that [Ca(2+)](i) oscillations are required to activate freshly ovulated oocytes, but if initiated at abnormally high frequency and duration or if induced in aged oocytes, the [Ca(2+)](i) oscillations may trigger premature termination of embryonic development.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.