Endostatin inhibits bradykinin-induced cardiac contraction

.Endogenous fragments of extracellular matrix are known to possess various biological effects. Levels of endostatin, a fragment of collagen type XVIII, increase in certain cardiac diseases, such as cardiac hypertrophy and myocardial infarction. However, the influence of endostatin on cardiac contraction has not been clarified. In the present study, we investigated the effects of endostatin on bradykinin-induced atrial contraction. Isometric contractile force of mouse isolated left atria induced by electrical current pulse was measured. Voltage-dependent calcium current of guinea pig ventricular myocytes was measured by a whole-cell patch-clamp technique. Endostatin (100–1,000 ng/ml) alone treatment had no influence on left atrial contraction. On the other hand, pretreatment with endostatin (300 ng/ml) significantly inhibited bradykinin (1 µM)-induced contraction and voltage-dependent calcium current. These data suggest that endostatin may decrease bradykinin-induced cardiac contraction perhaps through the inhibition of voltage-dependent calcium channel.     

Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency

ObjectiveTo examine the presence and effect of calstabin2-deficiency in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy (ARVC).AnimalsThirteen Boxer dogs with ARVC.Materials and methodsTissue samples were collected for histopathology, oligonucleotide microarray, PCR, immunoelectrophoresis, ryanodine channel immunoprecipitation and single-channel recordings, and calstabin2 DNA sequencing.ResultsIn cardiomyopathic Boxer dogs, myocardial calstabin2 mRNA and protein were significantly decreased as compared to healthy control dogs (calstabin2 protein normalized to tetrameric cardiac ryanodine receptor (RyR2) complex: affected, 0.51 ± 0.04; control, 3.81 ± 0.22; P < 0.0001). Calstabin2 deficiency in diseased dog hearts was associated with a significantly increased open probability of single RyR2 channels indicating intracellular Ca²⁺ leak. PCR-based sequencing of the promoter, exonic and splice site regions of the canine calstabin2 gene did not identify any causative mutations.ConclusionsCalstabin2 deficiency is a potential mechanism of Ca²⁺ leak-induced ventricular arrhythmias and heart disease in Boxer dogs with ARVC.     

Dopamine Release Suppression Dependent on an Increase of Intracellular Ca2+ Contributed to Rotenone-induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson’s disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca²⁺ dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca²⁺ in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca²⁺ through Ca²⁺ influx by opening of the voltage-gated Ca²⁺ channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP₂) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca²⁺ channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca²⁺ was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.     

A single injection of periostin decreases cardiac voltage-gated Na+ channel in rat ventricles

Changes in electrophysiological properties, such as ion channel expression and activity, are closely related to arrhythmogenesis during heart failure (HF). However, a causative factor for the electrical remodeling in HF has not been determined. Periostin (POSTN), a matricellular protein, is increased in heart tissues of patients with HF. In the present study, we investigated whether a single injection of POSTN affects the electrophysiological properties in rat ventricles. After male Wistar rats were intravenously injected with recombinant rat POSTN (64 µg/kg, 24 hr), electrocardiogram (ECG) was recorded. Whole-cell patch clamp was performed to measure action potential (AP) and Na⁺ current (I_Na) in isolated ventricular myocytes. Protein expression of cardiac voltage-gated Na⁺ channel (Na_V1.5) in isolated ventricles was examined by Western blotting. In ECG, POSTN-injection significantly increased RS height. POSTN-injection significantly delayed time to peak in AP and decreased I_Na in the isolated ventricular myocytes. POSTN-injection decreased Na_V1.5 expression in the isolated ventricles. It was confirmed that POSTN (1 µg/ml, 24 hr) decreased I_Na and Na_V1.5 protein expression in neonatal rat ventricular myocytes. This study for the first time demonstrated that a single injection of POSTN in rats decreased I_Na by suppressing Na_V1.5 expression in the ventricular myocytes, which was accompanied by a prolongation of time to peak in AP and an increase of RS height in ECG.     

Differential expression and localization of TRPV channels in the mature sperm of Anas platyrhynchos

Sperm cells perform precise chemotactic and thermotactic movement which is crucial for fertilization. However, the key molecules involved in detection of different chemical and physical stimuli which guide the sperm during navigation are not well understood. Ca²⁺-signalling mediated by ion channels seem to play important role in motility and other fertility parameters. In this work, we explored the endogenous localization pattern of TRPV channels in the mature spermatozoa of avian species. Using sperm from white pekin duck (Anas platyrhynchos) as the representative avian model, we demonstrate that duck sperm endogenously express the thermosensitive channels TRPV1, TRPV2, TRPV3, TRPV4, and highly Ca²⁺-selective channels TRPV5 and TRPV6 in specific yet differential locations. All of these TRPV channels are enriched in the sperm tail, indicating their relevance in sperm motility. Interestingly, the TRPV3 and TRPV4 channels are present in the mitochondrial region. Calcium selective TRPV5 channel is exclusively present in sperm tail and is most abundant among the TRPV channels. This is the first report describing the endogenous presence of TRPV2 and TRPV3 channels in the sperm of any species. Using confocal imaging and super-resolution imaging, we demonstrate that though the TRPV channels are evolutionarily closely related, they have distinct localization pattern in the duck sperm, which could impact their role in fertilization.     

Inhibitory effects of SKF96365 on the activities of K+ channels in mouse small intestinal smooth muscle cells

In order to investigate the effects of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), which is a non-selective cationic channel blocker, on K⁺ channel currents, we recorded currents through ATP sensitive K⁺ (I_KATP), voltage-gated K⁺ (I_Kv) and Ca²⁺ activated K⁺ channels (I_BK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished I_KATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K⁺ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC₅₀) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC₅₀ value in I_KATP suppression) reversibly inhibited the I_Kv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient I_BK and caffeine-induced I_BK. These results indicated that SKF inhibited ATP sensitive K⁺ channels and voltage-gated K⁺ channels, with the ATP sensitive K⁺ channels being more sensitive than the voltage-gated K⁺ channels. These inhibitory effects on K⁺ channels should be considered when SKF is used as a cationic channel blocker.     

Arginine promotes myogenic differentiation and myotube formation through the elevation of cytoplasmic calcium concentration

This study aimed to explore the mechanism underlying arginine-promoted myogenesis of myoblasts. C2C12 cells were cultured with a medium containing 0.1, 0.4, 0.8, or 1.2 mmol/L arginine, respectively. Cell proliferation, viability, differentiation indexes, cytoplasmic Ca²⁺ concentration, and relative mRNA expression levels of myogenic regulatory factors (MRF) and key Ca²⁺ channels were measured in the absence or presence of 2 chemical inhibitors, dantrolene (DAN, 10 μmol/L) and nisoldipine (NIS, 10 μmol/L), respectively. Results demonstrated that arginine promoted myogenic differentiation and myotube formation. Compared with the control (0.4 mmol/L arginine), 1.2 mmol/L arginine upregulated the relative mRNA expression levels of myogenin (MyoG) and Myomaker at d 2 during myogenic induction (P < 0.05). Cytoplasmic Ca²⁺ concentrations were significantly elevated by arginine supplementation at d 2 and 4 (P < 0.05). Relative mRNA expression levels of Ca²⁺ channels including the type 1 ryanodine receptor (RyR1) and voltage-gated Ca²⁺ channel (Cav1.1) were upregulated by 1.2 mmol/L arginine during 2-d myogenic induction (P < 0.01). However, arginine-promoted myogenic potential of myoblasts was remarkably compromised by DAN and NIS, respectively (P < 0.05). These findings evidenced that the supplementation of arginine promoted myogenic differentiation and myotube formation through increasing cytoplasmic Ca²⁺ concentration from both extracellular and sarcoplasmic reticulum Ca²⁺.     

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca²⁺ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca²⁺ concentration [Ca²⁺]_i and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca²⁺]_i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca²⁺ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca²⁺ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca²⁺ ([Ca²⁺]i): transient elevations in [Ca²⁺]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca²⁺ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca²⁺]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca²⁺ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca²⁺]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca²⁺ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca²⁺]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca²⁺ wave and spiral-like Ca²⁺ oscillations, respectively.     

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2α synthesis in bovine endometrium: role of calcium

The objective of these experiments was to determine the role of Ca²⁺ during oxytocin-stimulated prostaglandin (PG) F_2α release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF_2α release. A23187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF_2α in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca²⁺ in the cytoplasm by inhibiting endoplasmic reticulum Ca²⁺/ATPase pumps) stimulated release of PGF_2α in a concentration-dependent manner as well (P < 0.13). Oxytocin (10⁻⁶ M), AlF₄⁻ (a nonspecific activator of G-proteins; 10⁻⁵ M), A23187 (10⁻⁵ M), and melittin (a stimulator of phospholipase A₂; 10⁻⁴ M) stimulated PGF_2α release when explants were incubated in Ca²⁺-free medium (P < 0.10); however, oxytocin, A23187, or melittin were unable to stimulate PGF_2α release when explants were incubated in Ca²⁺-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AlF₄⁻ from stimulating phospholipase C activity (P < 0.08). CoCl₂ (a nonspecific Ca²⁺ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca²⁺ channel blocker) prevented oxytocin from stimulating PGF_2α release (P < 0.05). Our results suggest that both extracellular and intracellular Ca²⁺ may be required for oxytocin to stimulate PGF_2α secretion in bovine endometrial tissue.     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

Transient receptor proteins illuminated: Current views on TRPs and disease

The transient receptor potential proteins (TRPs) make up a very important family of ion channels responsible for a wide array of cellular functions. Originally identified in the visual system of Drosophila melanogaster, these channels are ubiquitously distributed throughout the mammalian system. The TRP family is divided into seven subfamilies in two groups: the first group comprises TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPN (Drosophila NOMPC) and TRPA (ankyrin). The second group contains TRPML (mucolipin) and TRPP (polycystin).The biophysical characterization of TRPs has revealed significantly different activation mechanisms and selectivity between channels. Functional studies have demonstrated that TRPs are necessary for a number of physiological processes, including sensation (such as taste, smell and temperature), hormone secretion and development. TRPs mediate these effects mainly by controlling the intracellular Ca²⁺ concentration, which acts as a second messenger. Recent research has linked TRPs to different diseases. This review considers the impact of TRPs on cell physiology and the abnormalities observed with channel dysfunction.     

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca²⁺ concentration ((Ca²⁺)_i), the present study aimed to determine the changes in Ca²⁺ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca²⁺ store of freshly prepared milk cells was estimated from the elevation of (Ca²⁺)_i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca²⁺ store in milk cells after intracellular Ca²⁺ depletion by Bapta-AM followed by spiking with 2.5 mM Ca²⁺ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca²⁺ store in milk cells was much less than blood PMNs. The production of superoxide anion (O₂^?) in vitro in response to (Ca²⁺)_i-dependent or (Ca²⁺)_i-independent modulators was used to evaluate the relevance of altered Ca²⁺ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O₂^? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca²⁺ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca²⁺ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.     

Use of intravenous lidocaine to prevent reperfusion injury and subsequent multiple organ dysfunction syndrome

Objective: The objective of this article is to review the human and veterinary literature and provide evidence for the potential beneficial effects of intravenous (IV) lidocaine hydrochloride in preventing post‐ischemic–reperfusion injury, the systemic inflammatory response syndrome (SIRS), and subsequent multiple organ dysfunction syndrome (MODS). Human data synthesis: Lidocaine is a local anesthetic and antiarrhythmic agent that has been used for years in human and veterinary medicine for the treatment of ventricular dysrhythmias associated with blunt cardiac trauma, myocardial ischemia, and cardiac surgery. More recently, the drug has been touted as a scavenger of reactive oxygen species (ROS), and has been used to prevent reperfusion dysrhythmias after treatment of myocardial infarction, cross‐clamping of the aorta, and in trauma medicine. Veterinary data synthesis: Although no clinical experiments with prophylactic intravenous lidocaine exist in veterinary medicine, there is a large body of evidence from experimental animals that support the use of lidocaine as a Na⁺/Ca²⁺ channel blocker, superoxide and hydroxyl radical scavenger, inflammatory modulator, and potent inhibitor of granulocyte functions. Lidocaine is being used in some clinical situations in an attempt to prevent the SIRS in veterinary trauma patients. ^a,b, ^a,b Conclusions: A large body of experimental evidence exists supporting the use of lidocaine as an anti‐oxidant and inflammatory modulator useful in preventing reperfusion injury. With the lack of cost‐effective and safe treatments for reperfusion injury in veterinary and human trauma medicine, the use of IV lidocaine to prevent the ensuing inflammatory response and MODS makes it an attractive addition to existing treatments. Therefore, it is essential that prospective clinical trials involving lidocaine as a treatment for prevention of reperfusion injury be performed in companion animals to demonstrate its safety and efficacy.     

Polymorphism identification and cardiac gene expression analysis of the calsequestrin 2 gene in broiler chickens with sudden death syndrome

1. Sudden death syndrome (SDS) in broilers is a cardiac disease associated with ventricular tachycardia (VT) and ventricular fibrillation (VF); however, its pathogenesis at the molecular level is not precisely determined.

2. Downregulation and mutations of calsequestrin 2 (CASQ2), a major intracellular Ca²⁺ buffer, have been associated with VT and sudden cardiac death (SCD) in humans but in chickens there is no report describing CASQ2 abnormalities in cardiac diseases.

3. In order to better understand the molecular mechanisms predisposing the myocardium to fatal arrhythmia in broilers, the mRNA expression level of chicken CASQ2 gene (chCASQ2) in the left ventricle of dead broilers with SDS was determined and compared to healthy broilers using quantitative real-time PCR (qPCR). To determine the probable mutations in chCASQ2, PCR and direct sequencing were also done.

4. Results showed a reduction in chCASQ2 expression in broilers dead by SDS. Three novel mutations (K289R, P308S, D310H) which are absent in healthy broilers were observed in chCASQ2.

5. It is concluded that susceptibility to fatal cardiac arrhythmia in SDS may be associated with changes in intracellular Ca²⁺ balance due to mutation and downregulation of chCASQ2.

     

Dynamic analysis of Ca2+ level during bovine oocytes maturation and early embryonic development

Mammalian oocyte maturation and early embryo development processes are Ca²⁺-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca²⁺ and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca²⁺ was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca²⁺ was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca²⁺ was present throughout the blastomere. In PA embryos, Ca²⁺ was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca²⁺ in the SCNT embryos. However, Ca²⁺ was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca²⁺ showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca²⁺ location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.     

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca²⁺-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca²⁺]_i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca²⁺-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.