Pattern Of Calcium Oscillations Research Articles

Ionomycin-induced mouse oocyte activation can disrupt preimplantation embryo development through increased reactive oxygen species reaction and DNA damage

Oocyte activation induced by calcium oscillations is an important process in normal fertilization and subsequent embryogenesis. In the clinical-assisted reproduction, artificial oocyte activation (AOA) is an effective method to improve the clinical outcome of patients with null or low fertilization rate after ICSI. However, little is known about the effect of AOA on preimplantation embryo development in cases with normal fertilization by ICSI. Here, we used ionomycin at different concentrations to activate oocytes after ICSI with normal sperm and evaluated energy metabolism and preimplantation embryo development. We found that a high concentration of ionomycin increased the frequency and amplitude of calcium oscillation patterns, affecting the balance of mitochondrial energy metabolism, leading to increased reactive oxygen species (ROS) and decreased ATP. Eventually, it increases DNA damage and decreases blastocyst formation. In addition, the addition of vitamin C to the culture medium ameliorated the increase in ROS and DNA damage and rescued the abnormal embryo development caused by excessive ionomycin activation. This study provides a perspective that the improper application of AOA may have adverse effects on preimplantation embryo development. Thus, clinical AOA treatment should be cautiously administered.

Communicating with Mouse Oocytes via Regulating Calcium Oscillation Patterns by Nanosecond Pulsed Electric Fields

In the treatment of infertility, many methods for assisted oocyte activation have been proposed, yet each has drawbacks that could limit the efficiency of or introduce uncertainty to the activation. This study adopts a physical approach, using nanosecond pulsed electric fields (nsPEFs) to trigger repetitive calcium oscillations in mouse oocytes, which boosts the activation rate and improves embryo development. The underlying activation mechanism is unveiled via simulations based on a kinetic calcium-channel model. This effective, controllable technique may provide a better solution to male infertility.

A novel artificial oocyte activation method: using nanosecond pulsed electric fields (nsPEFs) to regulate calcium oscillation patterns

A novel artificial oocyte activation method: using nanosecond pulsed electric fields (nsPEFs) to regulate calcium oscillation patterns

Calcium homeostasis and low-frequency magnetic and electric field exposure: A systematic review and meta-analysis of in vitro studies

Low frequency magnetic field (LF MF) exposure is recurrently suggested to have the ability to induce health effects in society. Therefore, in vitro model systems are used to investigate biological effects of exposure. LF MF induced changes of the cellular calcium homeostasis are frequently hypothesised to be the possible target, but this hypothesis is both substantiated and rejected by numerous studies in literature. Despite the large amount of data, no systematic analysis of in vitro studies has been conducted to address the strength of evidence for an association between LF MF exposure and calcium homeostasis. Our systematic review, with inclusion of 42 studies, showed evidence for an association of LF MF with internal calcium concentrations and calcium oscillation patterns. The oscillation frequency increased, while the amplitude and the percentage of oscillating cells remained constant. The intracellular calcium concentration increased (SMD 0.351, 95% CI 0.126, 0.576). Subgroup analysis revealed heterogeneous effects associated with the exposure frequency, magnetic flux density and duration. Moreover, we found support for the presence of MF-sensitive cell types. Nevertheless, some of the included studies may introduce a great risk of bias as a result of uncontrolled or not reported exposure conditions, temperature ranges and ambient fields. In addition, mathematical calculations of the parasitic induced electric fields (IEFs) disclosed their association with increased intracellular calcium. Our results demonstrate that LF MF might influence the calcium homeostasis in cells in vitro, but the risk of bias and high heterogeneity (I2>75%) weakens the analyses. Therefore any potential clinical implications await further investigation.

Modelling mechanism of calcium oscillations in pancreatic acinar cells.

We present a simple model for calcium oscillations in the pancreatic acinar cells. This model is based on the calcium release from two receptors, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) through the process of calcium induced calcium release (CICR). In pancreatic acinar cells, when the Ca²⁺ concentration increases, the mitochondria uptake it very fast to restrict Ca(2+) response in the cell. Afterwards, a much slower release of Ca²⁺ from the mitochondria serves as a calcium supply in the cytosol which causes calcium oscillations. In this paper we discuss a possible mechanism for calcium oscillations based on the interplay among the three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosol. Our model predicts that calcium shuttling between ER and mitochondria is a pacemaker role in the generation of Ca²⁺ oscillations. We also consider the calcium dependent production and degradation of (1,4,5) inositol-trisphosphate (IP3), which is a key source of intracellular calcium oscillations in pancreatic acinar cells. In this study we are able to predict the different patterns of calcium oscillations in the cell from sinusoidal to raised-baseline, high frequency and low-frequency baseline spiking.

Sperm involved in recurrent partial hydatidiform moles cannot induce the normal pattern of calcium oscillations

To assess the Ca2+-releasing ability of sperm involved in partial hydatidiform moles. Analysis of the activating and Ca2+-releasing ability of human sperm. University hospital research laboratory. Patients undergoing intracytoplasmic sperm injection (ICSI) treatment. Microinjection of mouse and human oocytes with sperm. Measurement of the fertilizing and Ca2+-releasing ability of human sperm. The mouse oocyte Ca2+ analysis showed that only 19.0% (4/21) of the mouse oocytes injected with sperm involved in molar pregnancies exhibited a normal pattern of Ca2+ oscillations versus 63.2% (36/57) of those injected with control sperm. Further, 83.3% (15/18) of donated in vitro-matured human oocytes injected with deficient sperm did not exhibit any Ca2+ release, while 76.9% (10/13) failed to show normal pronuclear development. Yet the sperm oocyte activation factor phospholipase C zeta (PLCζ) was present in the majority (96.6%, n=113) of the analyzed sperm at a normal expression level. Eventually, fertilization failure was overcome with assisted oocyte activation in subsequent therapeutic ICSI cycles, which led to normal deliveries. Sperm that previously provoked recurrent partial hydatidiform mole pregnancies due to dispermic fertilization is not able to activate human oocytes or trigger the normal pattern of Ca2+ oscillations in mouse and human oocytes in vitro.

Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization

What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO(2). The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann-Whitney U-test. Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.

Spatial Wavelet Analysis of Calcium Oscillations in Developing Neurons

Calcium signals play a major role in the control of all key stages of neuronal development, and in particular in the growth and orientation of neuritic processes. These signals are characterized by high spatial compartmentalization, a property which has a strong relevance in the different roles of specific neuronal regions in information coding. In this context it is therefore important to understand the structural and functional basis of this spatial compartmentalization, and in particular whether the behavior at each compartment is merely a consequence of its specific geometry or the result of the spatial segregation of specific calcium influx/efflux mechanisms. Here we have developed a novel approach to separate geometrical from functional differences, regardless on the assumptions on the actual mechanisms involved in the generation of calcium signals. First, spatial indices are derived with a wavelet-theoretic approach which define a measure of the oscillations of cytosolic calcium concentration in specific regions of interests (ROIs) along a cell, in our case developing chick ciliary ganglion neurons. The resulting spatial profile demonstrates clearly that different ROIs along the neuron are characterized by specific patterns of calcium oscillations. Next we have investigated whether this inhomogeneity is due just to geometrical factors, namely the surface to volume ratio in the different subcompartments (e.g. soma vs. growth cone) or it depends on their specific biophysical properties. To this aim correlation functions are computed between the activity indices and the surface/volume ratio along the cell: the data thus obtained are validated by a statistical analysis on a dataset of different cells. This analysis shows that whereas in the soma calcium dynamics is highly correlated to the surface/volume ratio, correlations drop in the growth cone-neurite region, suggesting that in this latter case the key factor is the expression of specific mechanisms controlling calcium influx/efflux.

Roles of 3,4-methylenedioxymethamphetamine (MDMA)-induced alteration of connexin43 and intracellular Ca2+ oscillation in its cardiotoxicity

Although it is well known that 3,4-methylenedioxymethamphetamine (MDMA) can cause various cardiovascular abnormalities and even sudden death from cardiac arrhythmia, whether it has any effect on myocardial gap junctions, which might be one of the targets mediating MDMA-induced cardiotoxicity, remains unclear. ObjectiveTo test the hypothesis that MDMA may affect the myocardial gap junction protein connexin43 (Cx43) and induce cardiac dysrhythmia. Method(1) In vivo study: adult rats were treated with a single dose MDMA administration (20mg/kg, i.p.). Electrocardiogram detection and immunohistochemical analysis were performed to evaluate cardiac function and expression of Cx43, respectively; (2) in vitro study: cultured ventricular myocytes of neonatal rats were treated with MDMA (10, 100, 1000μmol/L) for 1h. Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to investigate the total Cx43 mRNA expression. Immunofluorescent analysis was used to evaluate the amount of junctional Cx43. The phosphorylation status of Cx43 at site Ser368 and intracellular Ca2+ oscillation were also studied. ResultsObvious changes in electrocardiographic patterns were found in rats following MDMA administration. They were characterized by prolonged QRS duration associated with increased amplitude of QRS complex. The heart rates in treated rats were significantly decreased compared to the rats in the control group. The immunohistochemical findings revealed a significant decrease in Cx43 expression. The in vitro study also showed a marked decline in total Cx43 protein associated with reduction of Cx43 mRNA, whereas the phosphorylated Cx43 at Ser368 was increased. Decrease of junctional Cx43 was found correlated with reduction in N-cadherin induced by high concentration of MDMA. Additionally, confocal microscopy findings revealed alteration of intracellular calcium oscillation patterns characterized by high frequency and increasing influx Ca2+. ConclusionsMDMA reduces expression of cardiac gap junction protein Cx43. The increase of phosphorylation status of Cx43 at Ser368 induced by MDMA is attributed, at least in part, to the Ca2+-dependent regulation of protein kinase C (PKC) activity. Our findings provide first evidence of MDMA-mediated changes in those cardiac gap junctions that may underlie MDMA-induced cardiac arrhythmia.

Characterization of calcium oscillation patterns in caprine oocytes induced by IVF or an activation technique used in nuclear transfer

Routine activation of nuclear transfer (NT) eggs involves the application of a single intracellular calcium [Ca 2+] i rise, stimulated by an electrical pulse, as opposed to [Ca 2+] i oscillations, which is the natural mode of sperm-induced activation at fertilization in all mammalian species tested to date. It has yet to be shown that caprine oocytes exhibit an increase in calcium at fertilization in a manner similar to other mammals. The objective of the present study was to evaluate and characterize the ([Ca 2+] i) oscillation patterns of caprine metaphase II (MII) oocytes during IVF and during an activation techniques used in nuclear transfer. Additionally, the effect of cytochalasin B (cyto B) in the NT process was evaluated for its impact on [Ca 2+] i oscillations and subsequent embryo development. Mature in vitro and in vivo derived caprine oocytes were activated by 5 μM ionomycin, an electrical pulse(s), or IVF. The intracellular Ca 2+ response was determined using the [Ca 2+] i indicator Fura-2 dextran (Fura-2D). Ova treated with ionomycin or stimulated by an electrical pulse exhibited a single [Ca 2+] i rise, whereas IVF-derived oocytes showed oscillations. IVF [Ca 2+] i showed some variation, with 62% of in vitro matured oocytes exhibiting oscillations, whereas 8% of in vivo matured oocytes exhibited oscillations demonstrating a correlation between [Ca 2+] i responses and maturation technique. Knowing the [Ca 2+] i profile of activated eggs, one may be able to optimize the activation methodology used in a production nuclear transfer setting which could potentially improve development to term for NT embryos.

Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice

[corrected] Transferring a germinal vesicle (GV) from an aged woman's oocyte into ooplasm from a younger woman has been proposed as a possible way to overcome the problem of age-related decline in female fertility. Here we assessed this possibility by determining whether ooplasts derived from young mice could rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. Three groups of reconstructed oocytes, young GV-young cytoplast (group YY), aged GV-young cytoplast (group AY), and young GV-aged cytoplast (group YA), were created by micromanipulation and electrofusion. Nuclear transplantation was successful in 89.8-94.4% of GV-ooplast complexes, and maturation rate of the reconstructed oocytes was 93.5-97.9%. Confocal microscopy analysis showed a significantly higher rate (49.2%) of chromosome misalignment in ageing mice than in young mice (16.9%), and 57.1% of oocytes in group AY exhibited chromosome misalignment, while the abnormality rate in groups YY and YA was 16.3 and 16.7% respectively. Calcium imaging showed that the three groups of reconstructed oocytes exhibited a similar pattern of calcium oscillations upon stimulation with bovine sperm extracts. Fertilization rate and developmental capacity to 2-cell embryos were also similar among the three groups of oocytes. Our findings suggest that: (i) the ooplasm from young mice could not rescue ageing-associated chromosome misalignment in meiosis of GV from aged mice; and (ii) behaviour of chromosome alignment over metaphase spindle is predominantly determined by GV material.

Cracking the calcium code

Cytosolic calcium has long been recognized as a second messenger for many intrinsic or environmental signals in animals and plants. In plant cells, cytosolic calcium elevations can be triggered by hormonal signals, such as abscisic acid (ABA) and auxins; abiotic environmental signals, such as cold or mechanical stimulation; and biotic signals involved in the recognition of pathogens or symbiotic organisms. It is intriguing that, although all these signals use cytosolic calcium as a second messenger, they give rise to different and sometimes even opposite physiological, biochemical or developmental responses. Thus, it has been hypothesized that the calcium signals involved have different features that enable the cells to use the same second messenger to generate distinct downstream responses. Indeed, calcium signals can differ in their amplitude, as well as in their spatial and temporal patterns. Recently, it was shown that calcium signals in Arabidopsis guard cells exhibit stimulus-specific patterns of oscillations and that an oscillating calcium signal is required for stomatal closure. However, until now there has been no evidence that a specific pattern of calcium oscillations is required to control a specific downstream physiological response.In a recent issue of Nature, Gethyn Allen et al. 1xA defined range of guard cell calcium oscillation parameters encodes stomatal movements. Allen, G.J. et al. Nature. 2001; 411: 1053–1057Crossref | PubMed | Scopus (340)See all References1 analyze the relationships between the temporal pattern of calcium oscillations in guard cells and the physiological response of stomatal closure in Arabidopsis. The authors generate cytosolic calcium oscillations with a set frequency and duration by repeatedly switching the guard cells under investigation rapidly from a low-calcium, depolarizing buffer to a high-calcium, hyperpolarizing buffer, to induce calcium influx. In guard cells engineered to express the GFP-based calcium indicator, cameleon, they monitor the stomatal aperture and the intracellular calcium concentration in parallel, observing that a single intracellular calcium elevation is sufficient to trigger fast stomatal closure. However, this ‘calcium-reactive’ closure can only be maintained and lead to long-term stomatal closure if the initial calcium elevation is followed by oscillations with a defined pattern. Using the calcium clamp-buffer system, the authors determine the number, duration and frequency parameters that are optimal to induce long-term ‘calcium programmed’ stomatal closure. Interestingly, these parameters fit with the parameters of calcium oscillations induced by ABA – a physiological signal that induces long-term stomatal closure.Moreover, the authors provide genetic evidence that the frequency and duration parameters of the oscillations are crucial for ABA-induced stomatal closure. They show that ABA triggers cytosolic calcium oscillations in guard cells of the ABA-insensitive mutant, gca2. However, the frequency of the oscillations is higher than in the wild type, the duration of the transients is much shorter and ABA fails to induce long-term ‘calcium programmed’ stomatal closure in this mutant. Nevertheless, long-term stomatal closure can be restored in the guard cells of gca2 by imposing calcium oscillations with a frequency and transient duration that cause long-term closure in the wild type.This study represents a major step towards understanding the specificity of calcium signaling in plant cells because it demonstrates a relationship between a defined pattern of cytosolic calcium oscillations and a downstream physiological response. It should trigger much interest in the analysis of the molecular machinery of calcium channels, pumps and transporters that generate endogenous calcium oscillations in plants, as well as the calcium-dependent pathways that decode the frequency and duration parameters of the calcium oscillations.

The Relationship between Pulsatile Secretion and Calcium Dynamics in Single, Living Gonadotropin-Releasing Hormone Neurons

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1–43 fluorescence to monitor changes of secretion in individual GT1–7 cells and then combined this approach with simultaneous measurement of intracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mm) or N-methyl-d-aspartate (NMDA, 100μ m) predictably evoked dynamic increases of both FM1–43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that[ Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons.

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1-43 fluorescence to monitor changes of secretion in individual GT1-7 cells and then combined this approach with simultaneous measurement ofintracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100 microM) predictably evoked dynamic increases of both FM1-43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that [Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.

Soluble extracts from ascidian spermatozoa trigger intracellular calcium release independently of the activation of the ADP ribose channel.

We have injected soluble extracts of sperm from the ascidian Ciona intestinalis into oocytes of the same species to test whether these extracts can mimic the events of fertilisation. Injection of ascidian sperm extracts leads, after a delay of approximately 60 s, to a large calcium transient and repetitive pattern of calcium oscillations, mimicking the normal fertilisation response. The response was concentration-independent, suggesting a stimulatory mechanism in triggering the fertilisation response. We tested the pathway of calcium release in ascidian oocytes after injection of sperm extracts by preinjection of calcium release inhibitors. The data demonstrate that dual pathways to calcium release act at fertilisation in ascidians, as in other species. C. intestinalis oocytes are characterised by a ion channel in the plasma membrane that is gated uniquely by ADP ribose. We show that this channel is not gated by the injection of ascidian sperm extracts. Our data suggest that one metabolic pathway triggered by sperm, the release of nitric oxide, is not stimulated by sperm extracts and that several metabolic pathways are stimulated at fertilisation by more than one factor within sperm.

Oocyte activation after intracytoplasmic injection of mature and immature sperm cells.

The inactivation of metaphase-promoting factor (MPF), leading to reactivation of the oocyte cell cycle after fertilization, is one of the most important results of oocyte activation in the case of intracytoplasmic injection of mature or immature sperm cells. Oocyte activation is a cell signalling event that is likely to imply receptors at the oocyte plasma membrane and a signal transduction pathway involving calcium and protein phosphorylation/dephosphorylation. The typical calcium signal during oocyte activation in mammals takes the form of calcium oscillations. Intracytoplasmic sperm injection (ICSI) is associated with a slightly different pattern of calcium oscillations in comparison with normal fertilization. Available data suggest that sperm cytosolic factor(s) play a role of oscillator, modulating the properties of the oocyte's intracellular calcium stores, whereas the role of trigger, normally realized by spermoocyte interactions at the level of their respective cell surfaces, can be supplemented in the conditions of ICSI by an artificial calcium influx generated by the procedure itself. Delayed onset and an abnormal form of the oocyte activation-promoting calcium signal are the two known molecular abnormalities of human oocyte activation; they can lead to fertilization failure and are also suspected to be at the origin of various embryo abnormalities. The impact of oocyte activation abnormalities on future development increases when oocytes are fertilized with immature sperm cells (spermatids) because the chromatin of these cells is less protected than sperm chromatin against a rapid action of the oocyte's MPF. If oocyte stimulation by the sperm calcium oscillation-promoting activity, which first appears at the round spermatid stage of human spermatogenesis, is insufficient to cause a rapid inactivation of MPF, premature condensation of spermatid chromatids may lead to aneuploidy.

Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes.

Among the possible mechanisms of oocyte activation after sperm penetration, it appears most likely that a protein released by the spermatozoon elicits a calcium elevation in the ooplasm. To further test this idea, cytosolic factors obtained from human spermatozoa by two different methods, freezing-thawing and sonication, were injected into mouse oocytes following which intracellular calcium release was measured. Of a total of 42 mouse oocytes, a pattern of calcium oscillations was observed in nine out of 16 oocytes injected with sonicated fraction, in all of eight oocytes with the frozen-thawed fraction and in none of 18 control oocytes. Injection of the frozen-thawed fraction also produced regular calcium oscillations in all of five in-vitro matured human oocytes. To assess the putative factor's ability to support fertilization, human oocytes that were not activated by prior intracytoplasmic injection of spermatozoa (ICSI) and round spermatids were reinjected with the frozen-thawed sperm fraction. Of 23 human oocytes which remained unfertilized after ICSI, 19 became activated after injection with sperm cytosolic factor; eight showed two pronuclei, three one pronucleus and eight showed three or more pronuclei. Of 11 oocytes unfertilized after prior round spermatid injection, two developed two pronuclei, four developed one pronucleus and two had three or more pronuclei. Cytogenetic analysis by fluorescence in-situ hybridization confirmed the existence of a male pronucleus in eight out of nine such zygotes displaying two or more pronuclei. Thus, human sperm extracts activated mouse and human oocytes after injection, as judged by calcium flux patterns in conjunction with male pronucleus formation.

Crosstalk between cellular morphology and calcium oscillation patterns Insights from a stochastic computer model

Agonist-induced oscillations in the concentration of intracellular free calcium ([Ca 2+] 1) display a wide variety of temporal and spatial patterns. In non-excitable cells, typical oscillatory patterns are somewhat cell-type specific and range from frequency-encoded, repetitive Ca 2+ spikes to oscillations that are more sinusoidal in shape. Although the response of a cell population, even to the same stimulus, is often extremely heterogeneous, the response of the same cell to successive exposures can be remarkably similar. We propose that such ‘Ca 2+ fingerprints’ can be a consequence of cell-specific morphological properties. The hypothesis is tested by means of a stochastic computer simulation of a two-dimensional model for oscillatory Ca 2+ waves which encompasses the basic elements of the two-pool oscillator introduced by Goldbeter et al. (Goldbeter A., Dupont G., Berridge M.J. Minimal model for signal-induced Ca 2+-oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–1465). In the framework of our extended spatiotemporal model, single cells can display various oscillation patterns which depend on the agonist dose, Ca 2+ diffusibility, and several morphological parameters. These are, for example, size and shape of the cell and the cell nucleus, the amount and distribution of Ca 2+ stores, and the subcellular location of the inositol(1,4,5)-trisphosphate-generating apparatus.