Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

Comparative study of preimplantation development following distinct assisted oocyte activation protocols in a PLC-zeta knockout mouse model.

Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI.

STUDY QUESTION: Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? SUMMARY ANSWER: ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. WHAT IS KNOWN ALREADY: Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. STUDY DESIGN, SIZE, DURATION: Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. MAIN RESULTS AND THE ROLE OF CHANCE: M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns. LIMITATIONS, REASONS FOR CAUTION: The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time. WIDER IMPLICATIONS OF THE FINDINGS: H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare.

Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure.

STUDY QUESTION: Does calcium oscillatory pattern analysis following heterologous intra-cytoplasmic sperm injection (ICSI) of human sperm into mouse oocytes lead to diagnostic and prognostic information for patients suffering from ICSI fertilization failure? SUMMARY ANSWER: We found that calcium oscillatory pattern analysis following heterologous ICSI has the strength to reveal, for the individual patient, the most probable underlying reason for low or failed fertilization after conventional ICSI. WHAT IS KNOWN ALREADY: Fertilization failure occurs in 1-3% of the couples undergoing conventional ICSI, for whom the mouse oocyte activation test (MOAT) or a similar heterologous ICSI model is the only diagnostic test available to evaluate the oocyte-activating capacity of human sperm cells. The MOAT classifies the patients into three groups: a low (group 1), an intermediate (group 2) and a high (group 3) activating group. In MOAT group 1 patients, a sperm-related deficiency is likely to be the cause of previous fertilization failures, while in MOAT group 3 patients a sperm-related deficiency can most probably be refuted. For MOAT group 2 patients, the result is called inconclusive; hence, both sperm and oocyte deficiencies may still contribute to the previous ICSI fertilization failure. STUDY DESIGN, SIZE, DURATION: The calcium-releasing ability of sperm from 26 MOAT patients with a history of zero or low fertilization following conventional ICSI was compared with the calcium-releasing ability of sperm from 4 control patients, with proven oocyte activation potential. Per case an average of 19 mouse oocytes were injected. Calcium imaging started within 5-10 min after ICSI and continued for 2 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm were demembranated with 0.02% lysolecithin for 1 min immediately before heterologous piezo-driven ICSI. For calcium imaging, metaphase II oocytes from B6D2/F1 mice were loaded with fura-2 acetoxymethyl ester. The calcium oscillatory patterns following heterologous ICSI were scored per oocyte and per patient individually based on the presence of calcium spikes and their frequency and amplitude. MAIN RESULTS AND THE ROLE OF CHANCE: For patients with low or high MOAT activating capacity (MOAT group 1 or 3, respectively), calcium analysis confirmed the MOAT result. For patients with a former inconclusive intermediate MOAT activating capacity result (MOAT group 2), no or strongly dissimilar calcium oscillatory patterns were seen, with significantly lower amplitude and frequency compared with control sperm. When the product of the amplitude and the frequency of the calcium traces was compared between the groups, MOAT group 1 and 2 cases differed significantly from MOAT group 3 cases and the control sperm (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The results of the calcium analysis in mouse oocytes should not be directly extrapolated to human oocytes, since it is well known that human spermatozoa exhibit a greater activating potency in mouse oocytes compared with mouse spermatozoa. Furthermore, not much is known yet about the influence of aberrant calcium oscillatory patterns, such as found in MOAT group 2 patients, on pre- and post-implantation embryo development in the human. WIDER IMPLICATIONS OF THE FINDINGS: Based on the current calcium oscillatory pattern analysis, we found that the product of calcium spike amplitude with its frequency allowed us to create a new threshold value, which can assist in confirming or refuting, on a single patient base, a sperm-borne activation deficiency. The latter is especially interesting for patients with a former intermediate inconclusive MOAT result (MOAT group 2 patients), for whom calcium oscillatory pattern analysis should be considered. STUDY FUNDING/COMPETING INTEREST(S): F.V.M. is holder of an aspirant clinical research mandate by the Flemish foundation of Scientific Research (FWO-Vlaanderen). B.H. is supported by a Ghent University grant (KAN-BOF E/01321/01). P.D.S. is holder of a fundamental clinical research mandate by the same Flemish foundation of Scientific Research (FWO-Vlaanderen).

Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

OBJECTIVES: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

A new look at osteoarthritis: Threshold potentials and an analogy to hypocalcemia.

Cartilage is a tissue that consist of very few cells embedded in a highly negatively charged extracellular matrix (ECM). This tissue is dealing with several electrical potentials which have been shown to control the production of ECM. Cartilage is present at joints and is constantly prone to degradation. Failing to repair the damage will result in the occurrence of osteoarthritis (OA). This perspective aims to link biophysical insights with biomolecular research in order to provide an alternative view on the possible causes of OA. Firstly, we hypothesize the existence of a threshold potential, which should be reached in order to initiate repair but if not met, unrepaired damage will evolve to OA. Measurements of the magnitude of this threshold electrical potential would be a helpful diagnostic tool. Secondly, since electrical potential alterations can induce chondrocytes to synthesize ECM, a cellular sensor must be present. We here propose an analogy to the hypocalcemia 'unshielding' situation to comprehend electrical potential generation and explore possible sensing mechanisms translating the electrical message into cellular responses. A better understanding of the cellular voltage sensors and down-stream signalling mechanisms may lead to the development of novel treatments for cartilage regeneration.

Gap26, a connexin mimetic peptide, inhibits currents carried by connexin43 hemichannels and gap junction channels.

Connexin mimetic peptides corresponding to short conserved extracellular loop sequences of connexins have been used widely as reversible inhibitors of gap junctional intercellular communication. These peptides also block movement of ATP and Ca(2+) across connexin hemichannels, i.e. hexameric channels yet to dock with partners in aligned cells and to generate the gap junction cell-cell conduit. By means of electrophysiology, we compared the effects of Gap26, a mimetic peptide corresponding to a short linear sequence in the first extracellular loop of connexin43, on connexin channel function in HeLa cells expressing connexin43. We demonstrate that Gap26 inhibited electrical coupling in cell pairs mediated by gap junctions after exposure for 30min. In contrast, Gap26 applied to single cells, inhibited hemichannel currents evoked in low Ca(2+) solution with a response time of less than 5min. The results further support the view that the likely primary and direct inhibitory effect of Gap26 is on connexin hemichannels, with gap junctions becoming inhibited later. The mechanism of action of Gap26 in blocking hemichannels and gap junction channels is discussed in the context of their different functions and locations.

Water-soluble CO-releasing molecules reduce the development of postoperative ileus via modulation of MAPK/HO-1 signalling and reduction of oxidative stress.

BACKGROUND AND AIMS: Treatment with carbon monoxide (CO) inhalation has been shown to ameliorate postoperative ileus (POI) in rodents and swine. The aim of this study was to investigate whether CO liberated from water-soluble CO-releasing molecules (CO-RMs) can protect against POI in mice and to elucidate the mechanisms involved. METHODS: Ileus was induced by surgical manipulation of the small intestine (IM). Intestinal contractility-transit was evaluated by video-fluorescence imaging. Leucocyte infiltration (myeloperoxidase), inflammatory parameters (ELISA), oxidative stress (lipid peroxidation), and haem oxygenase (HO)/inducible nitric oxide synthase (iNOS) enzyme activity were measured in the intestinal mucosa and muscularis propria. RESULTS: Intestinal contractility and transit were markedly restored when manipulated mice were pre-treated with CO-RMs. Intestinal leucocyte infiltration, expression levels of interleukin 6 (IL6), monocyte chemoattractant protein-1 and intercellular adhesion molecule-1, as well as iNOS activity were reduced by treatment with CORM-3 (a transition metal carbonyl that releases CO very rapidly); whereas expression of IL10/HO-1 was further increased when compared to nontreated manipulated mice. Moreover, treatment with CORM-3 markedly reduced oxidative stress and extracellular signal-related kinase (ERK)1/2 activation in both mucosa (early response) and muscularis (biphasic response). The p38 mitogen-activated protein kinase inhibitor SB203580 abolished CORM-3-mediated HO-1 induction. The HO inhibitor chromium mesoporphyrin only partially reversed the protective effects of CORM-3 on inflammation/oxidative stress in the muscularis, but completely abrogated CORM-3-mediated inhibition of the early "oxidative burst" in the mucosa. CONCLUSIONS: Pre-treatment with CO-RMs markedly reduced IM-induced intestinal muscularis inflammation. These protective effects are, at least in part, mediated through induction of HO-1, in a p38-dependent manner, as well as reduction of ERK1/2 activation. In addition, CORM-induced HO-1 induction reduces the early "oxidative burst" in the mucosa following IM.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

Intracellular free calcium related to apoptotic cell death in quail granulosa cell sheets kept in serum-free culture.

The relationship between apoptosis and resting intracellular free calcium ([Ca2+]i) was studied in serum-free cultures of granulosa cell sheets isolated from preovulatory quail follicles. Apoptosis was detected by acridine orange, in situ end-labeling of fragmented DNA and electron microscopy. [Ca2+]i was measured using fura-2. [Ca2+]i averaged 525 mM in freshly isolated sheets. In 24 h cultures no apoptosis was detected but [Ca2+]i became very dispersed, 20% of the sheets showing values above 1000 nM. At 48 h, apoptosis was obvious and [Ca2+]i remained dispersed. At 72 h, apoptosis and also the fraction of sheets with high [Ca2+]i were at their maximum. At 96 h apoptosis was subsiding and [Ca2+]i normalized. FSH depressed apoptosis and [Ca2+]i in the 72 h cultures. We conclude that at 24 h apoptosis is initiated at high [Ca2+]i foci. At later stages apoptosis is associated with high [Ca2+]i, but it is not clear whether this is cause or consequence.

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

Mitochondrial transmembrane potential changes support the concept of mitochondrial heterogeneity during apoptosis.

Dissipation of mitochondrial membrane potential (DeltaPsi(m)) and release of cytochrome c from mitochondria appear to be key events during apoptosis. The precise relationship (cause or consequence) between both is currently unclear. We previously showed in a model of serum-free cultured granulosa explants that cytochrome c is retained in a subset of respiring mitochondria until late in the apoptotic process. In this study we further investigated the issue of heterogeneity by using the DeltaPsi(m)-sensitive probe CM-H2TMRos in combination with a DNA fluorochrome. Changes of DeltaPsi(m) were assessed qualitatively by epifluorescence microscopy and were quantified using digital imaging microscopy. This approach yielded the following results: (a) CM-H2TMRos staining is a reliable and specific procedure to detect DeltaPsi(m) changes in granulosa cells explants; (b) dissipation of transmembrane potential is an early event during apoptosis preceding nuclear changes but is confined to a subpopulation of mitochondria within an individual cell; (c) in frankly apoptotic cells a few polarized mitochondria can be detected. These findings support the hypothesis that ATP needed for completion of the apoptotic cascade can be generated during apoptosis in a subset of respiring mitochondria and is not necessarily derived from anaerobic glycolysis.

ATP-dependent astrocyte-endothelial calcium signaling following mechanical damage to a single astrocyte in astrocyte-endothelial co-cultures.

In the brain, endfeet of perivascular astrocytes make close contact with capillary endothelial cells that form the blood-brain barrier. The aim of the present work was to investigate whether and how calcium signals can be communicated from astrocytes to endothelial cells following acute mechanical cell damage. The experiments were performed on astrocyte-endothelial co-cultures prepared from primary rat brain astrocytes and an endothelial cell line (ECV304). A single astrocyte was acutely damaged by mechanical stimulation of sufficient strength with a micropipette, and the resulting cytoplasmic calcium changes were monitored using fura-2 and digital calcium imaging. Mechanical damage to a single astrocyte triggered a large intercellular calcium wave that propagated to surrounding astrocytes and also to even remotely located (several hundred micrometers) endothelial cells. Astrocyte-endothelial calcium waves induced by mechanical cell damage were largely deflected by fast superfusion, were able to cross a cell-free lane, were dose-dependently inhibited by suramin, a P2-purinoceptor blocker, and were largely reduced in size in the presence of the ATP-degrading enzyme apyrase. Our results indicate that mechanical damage to a single astrocyte can produce far reaching calcium signals that are propagated by the release of a calcium mobilizing P2-purinergic agonist and that can be communicated to endothelial cells. As endothelial cytoplasmic calcium is an important factor in the regulation of blood-brain barrier permeability and transport, mechanical cell damage-induced astrocyte-endothelial calcium signals are hypothesized to play a role in the initiation of brain edema and the stimulation of brain glucose uptake.

Effect of flunarizine and methylprednisolone on functional recovery after experimental spinal injury.

The effect of flunarizine and methylprednisolone on the recovery of somatosensory evoked potentials (SEPs) was evaluated in an experimental model of spinal cord impact injury in anesthetized cats. In addition, the effect of flunarizine on posttraumatic spinal cord blood flow (SCBF) (using the hydrogen clearance technique) and interstitial calcium and potassium activity (ion-selective electrodes) was investigated. After the injury (600 g.cm), SEPs disappeared, followed by a spontaneous recovery to 17% of the preinjury amplitude at the end of the 4 h observation period. Flunarizine treatment (0.1 mg/kg IV, given 5 and 120 min after injury) resulted in a significantly improved recovery of SEPs, reaching 52% of the preinjury amplitude. Methylprednisolone treatment (30 mg/kg IV, given 5 min after injury) resulted in a 30% recovery level, significantly better than in untreated animals but significantly inferior to flunarizine treatment. Combination of both treatments resulted in a 62% recovery level, significantly better than after methylprednisolone treatment alone. Flunarizine treatment had no significant effect on the postinjury evolution of SCBF and interstitial potassium activity; it did, however, significantly accelerate the recovery of interstitial calcium activity, which sharply decreased immediately after injury. It is concluded that intravenous administration of the calcium entry blocker flunarizine improves the functional recovery of the spinal cord in the acute phase after experimental spinal impact injury. The observed improvement is not achieved by an effect on local blood flow but is possibly related to an inhibitory effect of the drug on cellular calcium entry.

First and second messenger role of calcium. Survival versus apoptosis in serum-free cultured granulosa explants.

In order to elucidate the causal relationship between increased intracellular free calcium ([Ca2+]i) and induction of apoptosis, serum-free cultured granulosa cell (GC) explants were subjected to various experimental protocols interfering with normal Ca2+ homeostasis. Modulation of apoptotic indices was calculated on DAPI-stained GC explants. In some experiments [Ca2+]i was measured with the Ca2+ probe fura-2 in combination with epifluorescence microscopy. Buffering of [Ca2+]i with BAPTA-AM resulted in inhibition of apoptosis, while increasing extracellular Ca2+ (otherwise called [Ca2+]e load) resulted in a biphasic response characterized by an initial inhibitory effect on apoptosis followed by a delayed phase of increased apoptosis that became apparent at 4 h after withdrawal of the [Ca2+]e load. The initial inhibitory effect of the [Ca2+]e load on apoptosis was dependent on the concentration of the load (range 2-50 mM), was augmented when the [Ca2+]e load was applied in the presence of the Ca2+ channel blocker methoxyverapamil, and was mimicked by applying Mg2+ and Gd3+, two Ca(2+)-receptor agonists. These observations point towards the involvement of an extracellular Ca(2+)-sensing receptor (CaR). Measurements of [Ca2+]i showed that the ion was increased just after [Ca2+]e load, followed by recovery that was complete at 2 h after the load. Collectively these data suggest that a [Ca2+]e load initiates apoptosis, becoming manifest 4 h later, by the provoked [Ca2+]i increase, and this effect is preceded by an apoptosis-inhibiting phase presumably involving CaR activation. We conclude that Ca2+ may act as a first (extracellular) messenger promoting cell survival and as a second (intracellular) messenger activating the cell death pathway.

Ionic changes accompanying astrocytic intercellular calcium waves triggered by mechanical cell damaging stimulation.

Mechanically poking or damaging a single cell within a confluent astrocyte culture produces the so-called intercellular calcium (Ca(2+)) waves, that is, cell-to-cell propagating changes of intracellular free Ca(2+). We were interested whether intercellular Ca(2+) waves are also associated with changes in other intra- or extracellular ions. To that purpose, we investigated spatiotemporal changes of intracellular Ca(2+) (Ca(i)2+), sodium (Na(i)+) and protons (H(i)+) in primary cultures of rat cortical astrocytes using microfluorescence imaging with fura-2, SBFI and BCECF, respectively; changes of extracellular potassium (K(e)+) were monitored with K(+)-sensitive microelectrodes. Mechanical damage to a single cell by stimulation with a piezo-electrically driven micropipette initiated intercellular Ca(2+) waves that propagated to about 160 microm away from the stimulation point. Na(i)(+) increases could be detected in cells located 2-3 cell diameters from the stimulated cell, acidification was observed 1-2 cell diameters away and Ke(+) increases were measured up to 75 microm away. Kinetic analysis suggests that the Na(i)(+) and H(i)(+) changes occur after, and thus secondary to the Ca(i)(2+) changes. In contrast, K(e)(+) changes occurred very fast, even before the Ca(i)(2+) changes, but their propagation speed was too fast to implicate them as a trigger of Ca(i)(2+) changes. As Na(i)(+) is an important regulator of glycolysis in astrocytes, we hypothesize that astrocytic Na(i)(+) changes in cells located remotely from a damaged cell might be a signal that activates glycolysis thereby producing more lactate that is transferred to the neurons and increases their energy potential to survive the inflicted damage.

Intracellular pH changes during zona drilling.

OBJECTIVE: To investigate whether zona drilling of oocytes, a technique used to locally disrupt the zona pellucida, causes important changes in intracellular pH. DESIGN AND METHODS: In a first set of experiments, intracellular pH was measured during zona drilling of mouse oocytes, using pH-sensitive microelectrodes. In a second set of experiments human oocytes that failed to fertilize were used to measure intracellular pH during drilling. In these cells intracellular pH was measured using microfluorimetry with the pH-sensitive dye 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein. RESULTS: In mouse oocytes intracellular pH dropped from 7.25 +/- 0.02 (mean +/- SEM) to 7.09 +/- 0.03 during zona drilling, followed by a recovery to pH 7.17 +/- 0.02 after 4 minutes (n = 20). In human oocytes, intracellular pH dropped over 0.36 +/- 0.10 pH units during drilling, followed by a recovery that was complete within 4 minutes (n = 14). CONCLUSION: Zona drilling is associated with a significant cytoplasmic acidification both in mouse and human oocytes. This effect is perhaps related to the high incidence of cytoplasmic degeneration after zona drilling of human oocytes.

Effects of flunarizine on induced calcium transients as measured in fura-2-loaded neurons of the rat dorsal root ganglion.

The effect of the calcium entry blocker flunarizine on a high-potassium induced increase of intracellular free calcium was studied. The experiments were done with neurons isolated from rat dorsal root ganglia and loaded with the calcium-sensitive dye fura-2. The increase of calcium induced by 60 mmol/l potassium was abolished after removal of extracellular calcium, was reversibly reduced by 50 mumol/l cadmium (76% inhibition), 50 mumol/l nickel (25% inhibition) and 10 mumol/l nifedipine (18% inhibition), and reversibly increased after removal of extracellular sodium (26% increase). The potassium induced increase of intracellular calcium is, therefore, mediated by transmembrane calcium influx, probably to a large extent through cadmium-sensitive calcium channels. Flunarizine (5 min incubation followed 1 min wash-out) reduced the amplitude of the high-potassium induced calcium increase in a dose-dependent manner (Kd = 370 +/- 100 nmol/l; mean +/- SEM; n = 8), causing complete inhibition at a concentration of 10 mumol/l in the majority of cells. Flunarizine (> or = 1 mumol/l) caused a reversible increase of the resting level of intracellular calcium in some cells, an effect which disappeared in the absence of extracellular calcium. The drug (1 mumol/l had no influence on the time course of recovery of intracellular calcium subsequent to a rise induced by high-potassium or by the calcium ionophore A23,187. It is concluded that flunarizine acts as an inhibitor of depolarization-mediated calcium influx. At a concentration of 1 mumol/l, the drug presumably has no effect on cellular calcium extrusion and/or sequestration mechanisms.

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl.

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP(3)R) via its BH4 domain, thereby suppressing IP(3)R Ca(2+)-flux properties and protecting against Ca(2+)-dependent apoptosis. Here, we directly compared IP(3)R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP(3)R nor inhibited IP(3)-induced Ca(2+) release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP(3)R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP(3)R binding and inhibition. This difference in IP(3)R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP(3)Rs. In agreement with the IP(3)R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP(3)R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP(3)Rs and Ca(2+)-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca(2+) signaling and apoptosis.