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.     

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.     

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.     

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.     

Expression of the zebrafish intermediate neurofilament Nestin in the developing nervous system and in neural proliferation zones at postembryonic stages

Background

At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca²⁺ ([Ca²⁺]_i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca²⁺]_i oscillations through the generation of inositol 1,4,5-triphosphate (IP₃), which causes Ca²⁺ release by binding to IP₃ receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation.

Results

Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca²⁺]_i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP₃ receptors.

Conclusion

Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca²⁺]_i transients, and to be activated after completion of maturation.     

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca²⁺]_i) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH₄Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at −80 mV, NH₄Cl triggered an inward current, the main component of which is a Ca²⁺-dependent chloride current. Calcium imaging confirmed that NH₄Cl provoked a [Ca²⁺]_i increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I_In). These currents were stimulated during external addition of NH₄Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH₄Cl and extracellular acidic medium, was shown to trigger a [Ca²⁺]_i increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH₄Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.     

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.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

Appearance of Depolarization- and Maitotoxin-Induced [Ca2+]i Elevation in Single LAN-1 Human Neuroblastoma Cells on Exposure to Retinoic Acid

Abstract: LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca²⁺]_i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca²⁺]_i elevation elicited by 55 mM K⁺. Maitotoxin, a putative activator of voltage-sensitive Ca²⁺ channels, did not evoke an elevation of [Ca²⁺]_i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca²⁺]_i when superfused in retinoic acid-treated cells. Both high K⁺- and maitotoxin-induced [Ca²⁺]_i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd³⁺, an inorganic Ca²⁺-entry blocker, and by the snail toxin ω-conotoxin GVIA, which interacts with the N sub-type of voltage-sensitive Ca²⁺ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel sub-type, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca²⁺]_i due to Ca²⁺ influx elicited by membrane depolarization. K⁺-induced [Ca²⁺]_i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K⁺-induced increase of [Ca²⁺]_i was still present. In conclusion, the results of the present study demonstrated that retinoic acid-induced differentiation of LAN-1 cells, which lack a high K⁺-evoked [Ca²⁺]_i increase in the undifferentiated state, induces the functional expression of an ω-conotoxin GVIA-sensitive, dihydropyridine-insensitive N-type voltage-sensitive Ca²⁺ channel that can be activated by maitotoxin and negatively modulated by protein kinase C.     

Delayed activation of plasma membrane Ca pump in human neutrophils

The interplay between Ca²⁺ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium ([Ca²⁺]_i) was studied in suspensions of human neutrophils loaded with the [Ca²⁺]_i indicator, Fura-2. To reveal Ca²⁺ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of [Ca²⁺]_i were caused exclusively by PM Ca²⁺ transporters. Following preincubation, the addition of CaCl₂ induced, mediated by ionomycin, a transient rise of [Ca²⁺]_i, a spike, eventually decreasing to an intermediary [Ca²⁺]_i level. The ATP-dependent decrease of [Ca²⁺]_i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na⁺-free medium, suggesting that neither activity of protein kinase C nor exchange was necessary for generation of the Ca²⁺ spike. In conclusion, the PM Ca²⁺ pump was responsible for the Ca²⁺ spike by responding to the rapid rise of [Ca²⁺]_i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular [Ca²⁺]_i spikes in general.     

Inactivation of histone H1 kinase by Ca2+ in rabbit oocytes

The present study investigated the role of intracellular Ca²⁺ (Ca²⁺_i) elevation on the inactivation of maturation promoting factor (MPF) in rabbit oocytes. The effects of the number of Ca²⁺ stimulations and of the amplitude of Ca²⁺_i elevation on the profile of histone H1 kinase activity were determined. A Ca²⁺ stimulation consisted of transferring mature oocytes from culture medium to 0.3 M mannitol containing 0.1–1.0 mM CaCl₂, and pulsing them at 1.25 kV/cm for 10 μsec, or microinjecting 2–8 mM CaCl₂ into the oocyte cytoplasm. The number of electrically-induced Ca²⁺ stimulations was varied, and amplitude of the Ca²⁺_i rise was controlled by altering Ca²⁺ concentration in the pulsing medium or the injection pipette. Ca²⁺_i concentration was determined with fura-2 dextran; oocytes were snap-frozen at indicated time points and assayed for H1 kinase activity. The activity was quantified by densitometry and expressed as a fraction of activity in nonstimulated oocytes. Electrically-mediated Ca²⁺_i rises inactivated H1 kinase in a manner dependent on the number of Ca²⁺ stimulations. A single Ca²⁺ stimulation inactivated H1 kinase to 30–40% of its initial activity. However, H1 kinase inactivation was only transient, regardless of the amplitude of the electrically- or injection-mediated Ca²⁺_i elevation. Increasing the number of Ca²⁺ stimulations helped to maintain H1 kinase activity at basal (pronuclear) levels. The results show the necessity of a threshold of Ca²⁺_i concentration to trigger MPF inactivation, and suggest a role for the extended period of time over which Ca²⁺_i oscillates at fertilization. © 1995 Wiley-Liss, Inc.     

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca²⁺ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca²⁺ concentration in cytosol ([Ca²⁺]_i) and nuclei ([Ca²⁺]_n). After calibration, [Ca²⁺]_n was constantly higher than [Ca²⁺]_i before and after the chelation of extracellular Ca²⁺ suggesting an active Ca²⁺ accumulation system on nuclear membrane. [Ca²⁺]_n was also constantly higher than [Ca²⁺]_i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca²⁺]_n and [Ca²⁺]_i was identical, and [Ca²⁺]_i gradient towards the nucleus and peripheral or central [Ca²⁺]_n rise was observed after these stimulations. From these results, [Ca²⁺]_n is not only regulated by the active Ca²⁺ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca²⁺]_n or [Ca²⁺]_i rise from cell to cell; single spike or oscillatory change of [Ca²⁺]_n and [Ca²⁺]_i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca²⁺, suggesting that FCS stimulated [Ca²⁺]_n and [Ca²⁺]_i rise solely depending on Ca²⁺ influx from extracellular medium. The higher concentration of [Ca²⁺]_n and heterogeneous [Ca²⁺]_n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc.     

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca²⁺] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca²⁺] 4.4 mg/dl); PBS^{CaMg free}/FCS (PBS without Ca²⁺ and Mg²⁺ + 20% FCS [Ca²⁺] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca²⁺] 3.2 mg/dl) and PBS^{CaMg free}/BSA (PBS without Ca²⁺ and Mg²⁺ +0.4% BSA, [Ca²⁺] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS^{CaMg free}/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS^{CaMg free}/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

BIM EL-mediated apoptosis in cumulus cells contributes to degenerative changes in aged porcine oocytes via a paracrine action

Whether cumulus cells (CCs) contribute to oocyte aging remains controversial; in that regard, little is known about biochemical processes of gene expression in CCs surrounding aged oocytes. The objective was to elucidate contributions of CCs to porcine oocyte aging and degeneration, apoptosis and BIM expression in CCs during oocyte aging in vitro. When culture of cumulus oocyte complexes (COCs) was prolonged (68 h, which resulted in 24 h of aging), the rate of blastocyst formation following electro-activation was lower than that of oocytes aged without CCs (2.6 ± 0.1 vs 13.5 ± 1.3%, mean ± SEM; P < 0.05). In addition, the presence of CCs significantly accelerated spontaneous fragmentation of oocytes following prolonged (92 h) culture. Apoptotic CCs were present in COCs cultured for 68 h, and the abundance of Bim mRNA in CCs progressively increased after 56 h of culture (P < 0.05). Based on immunofluorescence, BIM protein expression was up-regulated in CCs surrounding aged oocytes; furthermore, quantification (Western blot) of BIM_EL protein progressively increased after 56 h of culture. Lastly, in a series of experiments to elucidate the signal pathway, blocking gap junctions (with 1-octanol) during aging did not eliminate the effect of CCs on accelerating oocyte aging, but prolonged co-culture of denuded oocytes with COCs after in vitro maturation reduced blastocyst rate relative to culture of denuded oocytes aged alone (4.15 ± 0.1 vs 11.0 ± 0.7%, P < 0.05). We concluded that apoptotic CCs, in which BIM_EL up-regulation was involved, accelerated oocyte aging and degeneration in vitro via a paracrine action.     

Rise of intracellular Ca2+ level causes the decrease of cyclin B1 and Mos in the newt eggs at fertilization

Unfertilized eggs of the newt, Cynops pyrrhogaster, are arrested at the second meiotic metaphase, with activity of the M‐phase promoting factor (MPF) maintained at a high level. After fertilization, the eggs resume the cell cycle, and emit the second polar body. When the change in [Ca²⁺]_i in the fertilized eggs was monitored by aequorin, an early increase in [Ca²⁺]_i was observed 5–10 min after insemination and continued for about 30 sec. A late increase in [Ca²⁺]_i then occurred 10–15 min after fertilization and continued for 30–40 min. The injection of 1,2‐Bis (2 aminophenoxy) ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA) into unfertilized eggs inhibited reinitiation of the cell cycle after fertilization. Western blot analysis with antibodies against cyclin B1 or Mos indicated that both cyclin B1 and Mos were present in unfertilized eggs, but both disappeared within 30 min after fertilization. Treatment with Ca²⁺‐ionophore decreased both cyclin B1 and Mos. Chymotryptic activity in Cynops egg extracts was not significantly increased after fertilization or activation by treatment with the Ca²⁺‐ionophore. No change in [Ca²⁺]_i was observed following treatment with cycloheximide, but the amount of both cyclin B1 and Mos rapidly decreased. These results indicate that resumption of meiosis in Cynops eggs is induced by an increase in [Ca²⁺]_i at fertilization, which causes degradation of both cyclin B1 and Mos by inhibition of de novo synthesis of those proteins. Mol. Reprod. Dev. 53:341–349, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. In Ca²⁺-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca²⁺]_i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca²⁺]_i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca²⁺]_i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca²⁺]_i rises by stimulating estrogen receptors leading to Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca²⁺-dependent pathway.     

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca²⁻]_i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca²⁻]_i and intracellular pH (pH_i) in Na⁺-containing and Na⁺-deficient bicarbonate/CO₂-buffered media to investigate the role of Na⁺ in these P-initiated changes. We found that P failed to initiate the AR in Na⁺-deficient medium, and that the initial rise in [Ca²⁺]_i following P (1 μg/ml) stimulation was similar for both media; however, the [Ca²⁺]_i in the Na⁺-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca²⁺]_i. Moreover, the differences in plateau [Ca²⁺]_i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca²⁺]_i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca²⁺]_i and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na⁺-dependent mechanism is important in the P-initiated human sperm AR. Such a Na⁺ requirement may reflect the involvement of this ion in pH_i regulation, as capacitated sperm that were incubated in a Na⁺-deficient medium for ≥ 30 min displayed a significantly lower pH_i. © 1996 Wiley-Liss, Inc.