Calcium ionophore improves embryonic development and pregnancy outcomes in patients with previous developmental problems in ICSI cycles.

BACKGROUND: Calcium (Ca2+) ionophores are now mainly considered as efficient treatments for fertilization failure. Recently, its application for rescuing poor embryo development was proposed but still non-routine. This study aimed to explore whether Ca2+ ionophore improves embryo development and pregnancy outcomes in patients with poor embryo development in previous intracytoplasmic sperm injection (ICSI) cycles. METHODS: This study included 97 patients undergoing assisted oocyte activation (AOA) with Ca2+ ionophore (calcimycin, A23187) treatment. Preimplantation embryonic development and clinical outcomes were compared between ICSI-AOA cycles (AOA group) and previous ICSI cycles of the same patients in which poor embryo developmental potential was present (non-AOA group). Subgroups stratified by maternal age (< 35, 35-40, ≥ 40 years, respectively) were analyzed separately. RESULTS: A total of 642 MII oocytes were collected in AOA group, and 689 in non-AOA group. Significantly higher day 3 good quality embryo rate (P = 0.034), good quality blastocyst formation rate (P <  0.001), and utilization rate (P <  0.001) were seen in AOA group. Similar results were seen in each subgroup. For pregnancy outcomes, there were significant differences in clinical pregnancy rate (P = 0.039) and live birth rate (P = 0.045) in total group. In subgroup aged < 35 years, biochemical (P = 0.038), clinical (P = 0.041), and ongoing pregnancy rate (P = 0.037) in AOA group were significantly higher than that in non-AOA group. No significant improvement for clinical outcomes for subgroups aged 35-40 and aged ≥40. CONCLUSION: The study suggests that calcimycin could improve preimplantation development and pregnancy outcomes in patients aged < 35 years with embryo developmental problems in previous ICSI cycles.

A retrospective analysis of artificial oocyte activation in patients with low or no fertilisation in intracytoplasmic sperm injection cycles.

Intracytoplasmic sperm injection (ICSI) is commonly used to treat severe male factor infertility in assisted reproduction. A small percentage of patients face suboptimal fertilisation rate or even fertilisation failure despite having ICSI. Artificial oocyte activation (AOA) has been proposed as a suitable method to overcome their problem. This is a retrospective cohort analysis of ICSI cycles undergoing AOA. Injected metaphase II oocytes were exposed to either calcium ionophore (A23187) after ICSI or injection of calcium chloride during ICSI followed by incubation with A23187 after ICSI. The previous ICSI cycles of the patients formed the historical control group. Thirty-four AOA cycles were analysed. The normal fertilisation rate (52.1%) was significantly improved in the AOA group. The percentage of failed fertilisation cycles (11.8%) were significantly reduced in the AOA group. The cumulative clinical pregnancy rate (47.1%) and live birth rate (29.4%) were significantly increased when compared to the previous cycles. Subgroup analysis revealed that the performance of the A23187 only protocol and the concomitant injection of calcium chloride protocol were comparable in terms of laboratory parameters and pregnancy outcomes. AOA is an effective method to improve the fertilisation rate and pregnancy outcome of infertile couples with previous fertilisation problem after ICSI.IMPACT STATEMENTWhat is already known on this subject? A failed and low fertilisation rate after ICSI is not uncommon in assisted reproduction. AOA is normally used to improve fertilisation but there are discrepancies in the efficacy of the treatment.What do the results of this study add? AOA improves the fertilisation rate and pregnancy outcomes of couples with suboptimal fertilisation rate and fertilisation failure in previous ICSI cycles. The efficacies of two AOA protocols were comparable. The A23187 only protocol was recommended because of its simplicity.What are the implications of these findings for clinical practice and/or further research? AOA should be considered as a routine procedure for infertile couples with compromised fertilisation rates in previous ICSI cycles.

Artificial oocyte activation to improve reproductive outcomes in couples with various causes of infertility: a retrospective cohort study.

RESEARCH QUESTION: Does calcium ionophore treatment of oocytes improve fertilization rate, embryo development and outcomes in specific groups of infertile couples? DESIGN: This retrospective cohort study involved 796 couples undergoing oocyte activation with calcium ionophore (A23187) after intracytoplasmic sperm injection (ICSI) between 2016 and 2018. All metaphase II oocytes were exposed to 5 µmol/l ionophore for 15 min immediately after ICSI, cultured in vitro to the blastocyst stage, and transferred to the uteri of recipients on day 5 or cryopreserved for transfer in the next cycle. The previous cycles of the same patients formed the control group. RESULTS: Among 1261 ICSI cycles and 796 ICSI-artificial oocyte activation (ICSI-AOA) cycles, implantation, positive beta-HCG, clinical pregnancy and live birth rates were significantly (P < 0.05 to P < 0.001) improved for all groups, compared with previous cycles, except live birth rate in women with primary ovarian insufficiency (POI). Compared with previous cycles, rates of blastulation (all P < 0.001) and high-quality blastocysts (P < 0.05 to P < 0.001) were increased significantly for couples with male factor (oligoasthenoteratozoospermia [OAT]), unexplained infertility and couples with both factors in the ICSI-AOA cycles. High-quality blastocyst rate was increased in couples with polycystic ovary syndrome (PCOS) (P = 0.0453). Miscarriage rates were decreased significantly (P < 0.05 to P < 0.001) in couples with OAT, PCOS and unexplained infertility in the treatment cycles. No significant differences were found for fertilization rate, embryo development or live birth rate in patients with POI between both groups. CONCLUSIONS: Artificial oocyte activation was able to 'rescue' the poor reproductive outcomes in certain types of infertile couples with history of failure to achieve pregnancy.

Pregnancy and neonatal outcomes of artificial oocyte activation in patients undergoing frozen-thawed embryo transfer: a 6-year population-based retrospective study.

PURPOSE: To evaluate the impact of artificial oocyte activation (AOA) in pregnancy and neonatal outcomes in infertile patients undergoing cryopreserved embryo transfer. METHOD: This retrospective study included 5686 patients' transferred embryos from routine intracytoplasmic sperm injection (ICSI) and 194 patients' transferred embryos from ICSI combined with AOA (ICSI-AOA) from January 2011 to December 2016. Pregnancy and neonatal outcomes of couples undergoing routine ICSI or ICSI-AOA were analyzed before and after propensity score matching. Artificial oocyte activation was performed with ionomycin. RESULTS: The pregnancy outcomes showed no significant difference in the rates of biochemical pregnancy, clinical pregnancy, implantation, miscarriage, ectopic pregnancy, multiple pregnancy, and live births between the routine ICSI and ICSI-AOA groups before and after propensity score matching, respectively. The assessment of neonatal outcomes showed no statistically significant differences in the birth defect rate, birth weight, gestational age, preterm birth rate, early-neonatal death rate, and fetal sex ratio between the two groups, and similar results were also observed in the two matched cohorts. CONCLUSION: Artificial oocyte activation with ionomycin does not adversely affect pregnancy and neonatal outcomes in patients undergoing frozen-thawed embryo transfer, which is beneficial to clinicians counseling patients on the risks of artificial oocyte activation.

Calcimycin Suppresses S100A4 Expression and Inhibits the Stimulatory Effect of Transforming Growth Factor ß1 on Keloid Fibroblasts.

Recent researches have indicated that S100A4 participates in tissue fibrosis, whereas calcimycin inhibits this process as a novel S100A4 transcription inhibitor. However, the relationship and mechanisms between calcimycin and S100A4 in keloid fibroblasts (KFs) remain unknown. The present research was aimed to evaluate the effect of calcimycin on S100A4 expression and pathogenesis in KFs. Keloid fibroblasts were cultured and exposed to different concentrations of calcimycin in the absence or presence of transforming growth factor ß1 (TGF-ß1). The results showed that the expression of S100A4 was significantly increased in keloid derived fibroblasts compared with normal skin fibroblasts. Calcimycin depressed S100A4 in a concentration- and time-dependent manner. Moreover, calcimycin suppressed TGF-ß1-induced collagen type I, fibronectin, and α-smooth muscle actin expression and cell viability in cultured KFs. Furthermore, calcimycin modulated expression of TGF-ß/Smad target genes Smad7 and phosphorylation of TGF-ß1-induced Smad2/3. This research for the first time confirmed the presence of S100A4 in KFs. Calcimycin inhibits the expression of S100A4, as well as KF proliferation and migration and extracellular matrix (ECM) synthesis. Taken together, these results indicate that calcimycin might be a therapeutic candidate to keloid or other related fibrotic disorders.

Mercurius solubilis attenuates scopolamine-induced memory deficits and enhances the motor coordination in mice.

AIM: The present study was designed to investigate the effect of mercurius solubilis (merc sol) on scopolamine induced memory deficits and motor coordination in mice. MATERIALS AND METHODS: Three different formulations of merc sol (30X, 200M, 1M) were screened for their in vitro antioxidant potential through DPPH (2, 2-diphenyl-1-picrylhydrazyl) and nitric oxide scavenging activity using response surface methodology. Memory impairment was induced by the administration of scopolamine (1mg/kg i.p.) for 3 days to mice and assessment of memory acquisition and retention was done using Morris water maze test, passive avoidance test, elevated plus maze test, light and dark box test, motor coordination was evaluated using rotarod test and inclined plan test. The involvement of ion channels and nitric oxide pathway in the observed effect of merc sol was elucidated by administration of veratrine (0.125 µg/kg, i.p.), A23187 (20 µg/kg, i.p.), L- arginine (40 mg/kg, i.p.), aminoguanidine (50 mg/kg, i.p.) 30 min prior to merc sol. Acute toxicity studies were performed in accordance with the OECD (Organisation for Economic Co-operation and Development) guidelines. RESULTS: In vitro studies have revealed merc sol 30 X to have maximum free radical and nitric oxide scavenging activity. Administration of merc sol 30 X to mice significantly reduced scopolamine induced memory deficits and motor incoordination in all the performance tasks. The calcium ionophore, A23187 significantly altered the effect of merc sol in mice. No major signs of toxicity were observed. CONCLUSION: Merc sol has antiamnesic effect in scopolamine induced deficits and motor coordination in mice.

Drug induced exocytosis of glycogen in Pompe disease.

Pompe disease is caused by a deficiency in the lysosomal enzyme α-glucosidase, and this leads to glycogen accumulation in the autolysosomes of patient cells. Glycogen storage material is exocytosed at a basal rate in cultured Pompe cells, with one study showing up to 80% is released under specific culture conditions. Critically, exocytosis induction may reduce glycogen storage in Pompe patients, providing the basis for a therapeutic strategy whereby stored glycogen is redirected to an extracellular location and subsequently degraded by circulating amylases. The focus of the current study was to identify compounds capable of inducing rapid glycogen exocytosis in cultured Pompe cells. Here, calcimycin, lysophosphatidylcholine and α-l-iduronidase each significantly increased glycogen exocytosis compared to vehicle-treated controls. The most effective compound, calcimycin, induced exocytosis through a Ca2+-dependent mechanism, although was unable to release a pool of vesicular glycogen larger than the calcimycin-induced exocytic pore. There was reduced glycogen release from Pompe compared to unaffected cells, primarily due to increased granule size in Pompe cells. Drug induced exocytosis therefore shows promise as a therapeutic approach for Pompe patients but strategies are required to enhance the release of large molecular weight glycogen granules.

[Application of calcium ionophore A23187 in ICSI for globozoospermia: A report of 2 cases and review of the literature].

OBJECTIVE: To investigate the pathogenesis of globozoospermia, fertilization ability of round-headed sperm, and the application value of assisted oocyte activation in intracytoplasmic sperm injection (ICSI) for the wives of glohozoospermia men. METHODS: We collected oocytes from the wives of 2 globozoospermia patients and randomly divided them into two groups after ICSI to receive calcium ionophore A23187-activation and conventional treatment, respectively. We reviewed the relevant literature published at home and abroad, and discussed the etiology of globozoospermia, fertilization ability of round-headed sperm, and treatment options for this disease. RESULTS: Quality embryos were obtained in the A23187-activation group while no fertilized oocytes, oocyte cleavage, quality embryos, or blastular formation were found in the conventional treatment group. Both women achieved pregnancy and gave birth to healthy neonates after transfer of the quality embryos from the A23187-activation group. CONCLUSION: Calcium ionophore A23187 can be applied to ICSI for the wives of globozoospermia men and bring about desirable clinical outcomes. Meanwhile, attention should be paid to its safety.

Ultrasound in clinical setting of secondary hyperparathyroidism.

Secondary hyperparathyroidism (sHPT) is one of the most common and serious complications of chronic kidney disease (CKD) and maintenance hemodialysis (MHD). In sHPT, the biology of parathyroid cells changes significantly toward diffuse and nodular hyperplasia. Diagnosis and treatment of sHPT are based on intact parathyroid hormone (i-PTH) serum levels and on the parameters of mineral metabolism. The morphological diagnosis of sHPT relies on 2 complementary imaging techniques: high-resolution ultrasonography with color Doppler imaging (US/CD) and 99mTc-methoxyisobutylisonitrile (MIBI) scintigraphy. The main objective of this review is to stimulate nephrologists to use US/CD of the parathyroid glands during the progression of CKD in order to aid clinical, pharmacological and surgical strategies. The primary role of US/CD in sHPT should be to integrate the clinical diagnosis by defining the number and volume of hyperplastic glands, although the international guidelines do not state when and why to perform US/CD. This review also evaluates the role of US/CD in clinical follow-up and assessment of therapeutic response of sHPT, and it highlights how US/CD can evaluate the effect of therapy with phosphate binders, vitamin D or its analogues and calcimimetics, which are changing the natural history of sHPT and the frequency of parathyroidectomy. Evaluation of the morphological and vascular changes of hyperplastic parathyroids is useful to guide percutaneous ethanol injection therapy and to support clinical, pharmacological and surgical strategies. Epidemiological studies are needed to establish how US/CD could change the management of sHPT and why it should be repeated in patients with high levels of serum i-PTH.

S100A4-induced cell motility and metastasis is restricted by the Wnt/ß-catenin pathway inhibitor calcimycin in colon cancer cells.

The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/ß-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced ß-catenin mRNA and protein levels despite the expression of Δ45-mutated ß-catenin. Consequently, calcimycin inhibited Wnt/ß-catenin pathway activity and the expression of prominent ß-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.

Inhibition of cervical cancer cell growth through activation of upstream kinases of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is a critical energy-balancing sensor in the regulation of cellular metabolism in response to external stimuli. Emerging evidence has suggested that AMPK is a potential therapeutic target for human cancers. AICAR, one of the pharmacological AMPK activators, has been widely used to suppress cancer cell growth through activation of LKB1, an upstream kinase of AMPK. However, frequent mutations and deletions of LKB1 found in some cancer cells limit the application of AICAR as an efficient therapeutic drug. Here we show that an alternative pharmacological AMPK activator, A23187, was able to inhibit cervical cancer cell growth through activation of Ca(2+)/calmodulin-dependent protein kinase kinase beta, another upstream kinase of AMPK. Using cervical cancer cell models, we found that HeLa (LKB1-deficient cell) responded less to the anti-proliferative effect exerted by AICAR treatment (p < 0.001) compared with CaSki and C41 (LKB1-expressing cells). Conversely, the anti-proliferative effect was increased significantly in HeLa but not in CaSki and C41 cells under treatment by A23187 (p < 0.001). Moreover, co-treatment of AICAR and A23187 was able to further enhance the inhibitory effect on cell growth of Hela, CaSki and C41 cells. Notably, both AICAR and A23187 exerted the anti-proliferative effect on cervical cancer cells by suppressing AMPK/mTOR signalling activity. These data suggest that A23187 could be an alternative potential therapeutic drug used for anti-proliferation in LKB1-deficient cancer cells.

Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa.

OBJECTIVE: To evaluate the effect of artificial oocyte activation (AOA) on intracytoplasmic sperm injection (ICSI) cycles using surgically retrieved sperm. DESIGN: Laboratory study. SETTING: Fertility/assisted fertilization center. PATIENT(S): Couples undergoing surgical sperm retrieval for ICSI (n = 204). INTERVENTION(S): Application of calcium ionophore A23187 for AOA. MAIN OUTCOME MEASURE(S): Cycles were divided into experimental groups according to the origin of the sperm used for injection and the type of azoospermia: [1] testicular sperm aspiration in nonobstructive-azoospermic patients (TESA-NOA group, n = 58), [2] TESA in obstructive-azoospermic patients (TESA-OA group, n = 48), [3] and percutaneous epididymal sperm aspiration in obstructive-azoospermic patients (PESA-OA, n = 98). For each experimental group, cycles where AOA was applied (subgroup: activation) were compared with cycles in which AOA was not applied (subgroup: control). The fertilization, high-quality embryo, implantation, and pregnancy rates were compared among the subgroups. RESULT(S): For patients undergoing TESA, AOA did not improve ICSI outcomes for either type of azoospermia. However, for cases in which the injected sperm were retrieved from the epididymis, a statistically significantly increased rate of high-quality embryos was observed with AOA. CONCLUSION(S): Artificial oocyte activation may improve ICSI outcomes in azoospermic patients when epididymal, but not testicular spermatozoa, are injected.

A birth from the transfer of a single vitrified-warmed blastocyst using intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.

OBJECTIVE: To present the effectiveness of diagnostic heterologous intracytoplasmic sperm injection (ICSI), mouse oocyte activation test (MOAT), and ICSI combined with assisted oocyte activation (AOA) in a globozoospermic patient. DESIGN: A case report. SETTING: A private IVF center, Japan. PATIENT(S): A patient with globozoospermia. INTERVENTION(S): MOAT in a mouse and ICSI combined with AOA in a human. MAIN OUTCOME MEASURE(S): Ultrastructure, MOAT, fertilization, and pregnancy. RESULT(S): The transmission electron micrographs showed 100% round-headed spermatozoa lacking an acrosome. MOAT showed that the fertilization rate was 68.4% (13/19) when AOA was used but 0% (0/19) when AOA was not used. After the diagnosis of globozoospermia and sperm-related activation deficiency, 17 human mature oocytes were activated with calcium ionophore after ICSI was performed. The fertilization rate was 88.2% (15/17), and 11 blastocysts were cryopreserved using the vitrification method to prevent severe ovarian hyperstimulation syndrome. A single vitrified-warmed blastocyst was transferred. A gestational sac with fetal heart movements was recognized, and a healthy boy weighing 3180 g was born at 40 weeks of gestation by cesarean section without any congenital abnormality. CONCLUSION(S): MOAT allows discrimination between sperm- and oocyte-related fertilization failures and shows the effectiveness of AOA.

Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles.

OBJECTIVE: To investigate the effect of calcium ionophore on the fertilization rate of a patient with normozoospermia who nonetheless exhibited a low fertilization rate in intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: In vitro fertilization center. PATIENT(S): A male patient whose sperm, though diagnosed as normal by semen analysis, exhibited a severely low fertilization rate in ICSI cycles. INTERVENTION(S): Oocytes were activated by calcium ionophore after ICSI. MAIN OUTCOME MEASURE(S): Fertilization rate after oocyte activation; ultrastructure and protein expression of the patient's sperm. RESULT(S): The fertilization rate of oocytes activated with calcium ionophore (12 of 15, 80.0%) was higher than that of the nonactivated oocytes (4 of 16, 25.0%). Four embryos derived from the activated oocytes were transferred, resulting in a twin pregnancy. Further investigation revealed abnormalities in the patient's sperm: many nuclear vacuoles were observed and the expression of some proteins was absent. CONCLUSION(S): Oocyte activation with calcium ionophore was effective at increasing the fertilization rate of dysfunctional sperm characterized by ultrastructural and protein expression anomalies.

Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report.

We report our attempts to achieve a successful pregnancy outcome with calcium ionophore A23187 and puromycin oocyte activation using sperm from a normozoospermic husband of a patient with previous repeated failed fertilization following ICSI. Oocytes from the female partner of a couple with a 4 year history of unexplained primary infertility with repeated failed fertilization following ICSI were used. In the latest ICSI attempt, oocytes were activated by treatment with calcium ionophore (5 min) and puromycin (5 h), then cultured. In this cycle, assisted oocyte activation with calcium ionophore and puromycin after ICSI resulted in a satisfactory fertilization rate (8/12; 66.7%); in prior cycles only one of 71 oocytes (1.4%) was fertilized. The outcome was a Caesarean section delivery of a healthy male infant without congenital abnormalities at 41 weeks, 2 days of gestation. In conclusion, the use of calcium ionophore and puromycin for oocyte activation was found to be a useful method in a case of repeated failed fertilization after ICSI.

Tissue factor de-encryption: ionophore treatment induces changes in tissue factor activity by phosphatidylserine-dependent and -independent mechanisms.

Coagulation is initiated on tissue-factor-bearing cells when factor VIIa complexes with membrane-bound tissue factor and activates factors X and IX. Cellular tissue factor activity does not correlate with tissue factor antigen; treatment with calcium ionophore rapidly increases tissue factor activity without increasing tissue factor antigen. Our study examined the effect of calcium ionophore A23187 on tissue factor activity of freshly isolated, lipopolysaccharide-stimulated monocytes and non-transformed human dermal fibroblasts. A23187 increased tissue factor activity on monocytes and fibroblasts in a dose-dependent fashion between 0.1 and 50 micromol/l ionophore. This increase in activity was proportional to an increase in intracellular calcium in monocytes. The increase in tissue factor activity was partially attributable to an increase in phosphatidylserine expression, as measured by increased prothrombinase activity (1.1- to 4-fold) on ionophore-treated cells. The phosphatidylserine-binding protein annexin V decreased tissue factor activity on both ionophore-treated and untreated cells, reflecting the role of phosphatidylserine in tissue factor activity. However, even in the presence of saturating concentrations of annexin V, the tissue factor activity of ionophore-treated cells was 1.3- to 11.3-fold higher than that of untreated cells, indicating that the increase in tissue factor activity did not result solely from increased expression of phosphatidylserine. A23187 increased tissue-factor-dependent activation of factors IX and X 1.4- to 7-fold on both cell types, indicating that ionophore treatment did not alter factor VIIa/tissue factor substrate specificity. We conclude that the mechanism by which calcium ionophore increases tissue factor activity is not unique to monocytoid or transformed cells. Furthermore, the ionophore-induced increase in activity is not solely the result of increased exposure to phosphatidylserine. Finally, tissue factor de-encryption by A23187 does not alter factor VIIa/tissue factor substrate specificity.

Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells.

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.

Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen.

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore-induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore-induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore.

OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

Magnesium attenuates the neutrophil respiratory burst in adult asthmatic patients.

INTRODUCTION: IV magnesium (Mg2+) has been proposed as an emergent treatment for acute asthma exacerbations. Recent studies have focused on the effects of Mg2+ on bronchial smooth muscle, yet asthma is primarily an inflammatory disease. OBJECTIVE: To assess the effects of Mg2+ on the neutrophil respiratory burst of adult patients with asthma. METHODS: A prospective, blind study of volunteer adult asthmatic patients was performed. The patients' polymorphonuclear neutrophils (PMNs) were isolated, purified, and placed into phosphate-buffered saline with the following test conditions: concentrations of magnesium chloride (MgCl2) added: 0 mmol MgCl2, 1 mmol MgCl2 (low), and 10 mmol MgCl2 (high) both with and without the calcium (Ca2+) ionophore A23187 (0.1 mmol). PMNs were activated using N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 mumol), and the production of superoxide (O2-) was measured by the spectrophotometric reduction of cytochrome c. RESULTS: Mg2+ reduced activated PMN O2- production compared with that for no Mg2+ (1.0 +/- 0.1 nmol O2-/5 x 10(5) PMN/min) in both low (-0.52* +/- 0.3 nmol O2-/5 x 10(5) PMN/min) and high (-0.76* +/- 0.3 nmol O2-/5 x 10(5) PMN/min; *p < 0.05) concentrations. The addition of A23187 increased O2- production in both the high (0.53* +/- 0.02 nmol O2-/5 x 10(5) PMN/min) and the low (1.5* +/- 0.6 nmol O2-/5 x 10(5) x 10(5) PMN/min) Mg2+ groups, with no change in the control group (1.2 +/- 0.2 nmol O2-/10(5) PMN/min). CONCLUSIONS: In clinically relevant concentrations, Mg2+ attenuates the neutrophil respiratory burst in adult asthmatic patients. Mg2+ appears to affect PMNs by interfering with extracellular Ca2+ influx. Mg2+ may have a beneficial anti-inflammatory effect in asthmatic individuals.