R-Phycoerythrin from Colaconema formosanum (Rhodophyta), an Anti-Allergic and Collagen Promoting Material for Cosmeceuticals

R-phycoerythrin (R-PE), a pigment complex found in red algae, was extracted and purified from a newly identified red alga, Colaconema formosanum, and its bioactivities were examined. It was revealed that R-PE treatment resulted in high cell viability (>70%) to the mammalian cell lines NIH-3T3, RBL-2H3, RAW264.7, and Hs68, and had no effect on cell morphology in NIH-3T3 cells. Its suppression effect was insignificant on the production of IL-6 and TNF-α in lipopolysaccharides-stimulated RAW264.7 cells. However, calcium ionophore A23187-induced β-hexosaminidase release was effectively inhibited in a dose-dependent manner in RBL-2H3 cells. Additionally, it was revealed to be non-irritating to bionic epidermal tissues. Notably, procollagen production was promoted in Hs68 cells. Overall, the data revealed that R-PE purified from C. formosanum exhibits anti-allergic and anti-aging bioactivities with no observed consequential toxicity on multiple mammalian cell lines as well as epidermal tissues, suggesting that this macromolecule is a novel material for potential cosmetic use.

3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

Calcium Ionophore (A23187) Rescues the Activation of Unfertilized Oocytes After Intracytoplasmic Sperm Injection and Chromosome Analysis of Blastocyst After Activation

Calcium is a crucial factor in regulating the biological behavior of cells. The imbalance of calcium homeostasis in cytoplasm will cause abnormal behavior of cells and the occurrence of diseases. In intracytoplasmic sperm injection (ICSI) cycle, the dysfunction of oocyte activation caused by insufficient release of Ca2+ from endoplasmic reticulum is one of the main reasons for repeated fertilization failure. Calcium ionophore (A23187) is a highly selective calcium ionophore, which can form stable complex with Ca2+ and pass through the cell membrane at will, effectively increasing intracellular Ca2+ levels. It has been reported that calcium ionophore (A23187) can activate oocytes and obtain normal embryos. However, there are few studies on unfertilized oocytes after calcium ionophore (A23187) rescue activation in ICSI cycle. The purpose of this study was to analyze the effects of calcium ionophore (A23187) rescue activation on the activation of unfertilized oocytes, embryonic development potential, embryonic development timing and chromosomal aneuploidy, and to compare and analyze the clinical data of patients with calcium ionophore (A23187) activation in clinical application. The results showed that a certain proportion of high-quality blastocysts with normal karyotype could be obtained after calcium ionophore (A23187) rescue activation of unfertilized oocytes, and it did not have a significant effect on the timing of embryo development. In clinical practice, direct activation with calcium ionophore (A23187) after ICSI was better than rescue activation the next day. In conclusions, the studies on the effectiveness and safety of calcium ionophore (A23187) rescue activation for oocytes with ICSI fertilization failure can enable some patients to obtain usable, high-quality embryos during the first ICSI cycle.

Changes in Red Blood Cell Membrane Properties: The Role of Metabolic Syndrome Components

Metabolic syndrome (MetS) is a cluster of metabolic, hormonal and hemodynamic disorders that contribute to a change in the structural and functional status of erythrocytes and contribute to dysregulation of their cation transport function, where Ca2+ -dependent potassium channels (KCa channels) play an important role. A MetS model was performed using male Wistar rats, which were divided into control and experimental groups. Rats in the control group were fed standard rat chow. Rats in the experimental group were exposed to a high-fat and high-carbohydrate (HFHC) diet for 12 weeks. The data obtained indicate that the HFHC diet led to obesity, high blood pressure, hyperglycemia, impaired glucose tolerance, and dyslipidemia. The level of glutathione (GSH) decreased in the erythrocytes of rats suffering from MetS, but the level of malondialdehyde (MDA) increased. It was shown that the amplitude of the membrane potential of erythrocytes of rats with MetS changed depending on the acting agent: when stimulated with calcium ionophore A23187 it decreased, when the redox system ascorbat –phenazine methosulfate was used, it increased compared to the control group. The data obtained indicate that a HFHC diet leads to changes in the physical and chemical properties of the erythrocyte membrane.

NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2⋅–), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2⋅– and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.

12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Leptin is involved in acrosome reaction by facilitating activation of MAPK cascades in the Chinese mitten crab, Eriocheir sinensis

The role of leptin has been documented in several studies, including activated threonine phosphorylation of extracellular signal-regulated kinase (ERK1/2) in the reproduction of rodents and humans. Our previous studies have demonstrated that mitogen-activated protein kinase (MAPK) cascades ERK, P38, and c-Jun N-terminal kinase (JNK) are involved in the spermatogenesis and acrosome reaction of Eriocheir sinensis. Therefore, the aim of this study was to investigate the expression of leptin and its receptor (LepR), and the effect of leptin on MAPK cascades during calcium ionophore A23187-induced spermatozoa acrosome reaction in crabs. Successful western blotting revealed a 16 kDa band for leptin, and 120 kDa and 90 kDa bands for the obese receptor (LepR), respectively, in the tested male reproductive tissues. Both leptin and LepR were localized at the pro-acrosomal vesicle and apical cap (AC) of spermatids, suggesting their role in the subsequent acrosome reaction. Moreover, acrosome reaction can be enhanced by leptin, and this effect decreased due to the anti-LepR antibody. Afterwards, we investigated the effects of leptin on MAPK cascades. The results showed that leptin mainly activated the phosphorylation of ERK, P38 and JNK proteins in the apical cap during the acrosome reaction in crab spermatozoa. This study addresses the role of leptin on spermatozoa, and suggests that leptin may induce molecular changes associated with spermatozoa during acrosome reaction.

112 The effect of biological extenders on invitro induction of the acrosome reaction in bovine spermatozoa

Biological extenders in semen are used to protect the integrity of the sperm cells during cryopreservation. The two most common extenders are egg yolk and milk, and initial experiments describing heparin capacitation for IVF were conducted with semen frozen in egg yolk extender. However, semen of many bulls used for IVF is frozen in milk-based extender, and it is not clear whether this affects the response to heparin capacitation. This study aims to assess whether the use of milk- or egg-based extenders in frozen-thawed bovine semen affects sperm's ability to undergo the acrosome reaction, utilising either the endogenous progesterone pathway or calcium ionophore A23187 to induce the acrosome reaction following heparin capacitation. Frozen semen from 12 bulls (6 using egg yolk citrate-based extender containing 20% egg yolk, and 6 using milk-based extender) was used for no less than 3 replicates each. All semen was commercially processed and cryopreserved using the same methodology. Semen was thawed and separated using a Percoll gradient and centrifugation at 400×g for 10min, then incubated for 4h in a capacitating medium containing heparin (10μgmL−1) at a concentration of 1×106 spermmL−1. Capacitated sperm were then introduced to progesterone (10μM) or A23187 (10 μM) for acrosome induction and stained with fluorescein isothiocyanate-conjugated peanut agglutinin (5μgmL−1). After treatment and staining, the sperm were analysed using flow cytometry to determine the percentage of populations with fluorescent acrosomes, indicating an exposed acrosome and an ongoing acrosome reaction. Fluorescent microscopy was also used to confirm fluorescence in acrosome-reacting samples. Results were analysed as a 2×3 factorial design by ANOVA with extender and acrosome induction treatment as factors. For egg yolk-extended semen, the mean percent of acrosome-reacted sperm was 83.7 (±6.8), 81.8 (±7), and 15.0 (±7.5) for sperm treated with progesterone, A23187, and heparin only, respectively. Compared to milk-extended semen, for which the mean percent acrosome-reacted was 15.0 (±8.2), 14.8 (±7.4), and 12.0 (±6.8) for progesterone, A23187, and heparin only, respectively. Significant differences were observed between extenders, with egg yolk-extended semen having a significantly greater response to acrosome reaction-inducing agents than semen extended with milk-based extenders (P<0.05). Also, it was observed that progesterone induced a similar percentage of acrosome reactions as A23187. Variation between bulls in the response to heparin capacitation has been observed and cannot be eliminated in this study. The difference observed between extenders warrants further study with a split ejaculate design. The possibility that the semen extender used during cryopreservation affects the response to heparin capacitation suggests that this should be accounted for in IVF protocols to increase the efficiency of this technology.