Characterization of Equine Phospholipase C Zeta: A Review and Preliminary Results on Expression Defects in Subfertile Stallions

In all mammalian species studied thus far, fertilization results in a series of intracellular Ca²⁺ ([Ca²⁺]_i) increases, referred to as oscillations, responsible for driving oocyte activation and embryonic development. Current evidence supports the notion that sperm-borne phospholipase C zeta (PLCZ) is responsible for the initiation of these [Ca²⁺]_i oscillations. Although this appears to be a highly conserved mechanism for oocyte activation, differences in PLCZ sequence, activity, and expression do exist among different species. Herein, we summarize the information supporting PLCZ as the oocyte-activating factor in mammals and present our current knowledge regarding the characterization of this protein in the horse. The equine sequence yielded a protein of high relative [Ca²⁺]_i-releasing activity. Equine PLCZ was expressed over the head region overlying the acrosome, equatorial segment, connecting piece between the head and midpiece, and on the principal piece of the flagellum of stallion sperm. Equine PLCZ expressed both over the head and tail sperm regions was catalytically active, with the latter representing a characteristic unique to the horse. We also present preliminary data in subfertile stallions displaying PLCZ expression defects, although further research is required to establish a clear association between these defects and fertility problems in the horse. In summary, the information presented raises the questions of whether equine PLCZ could play diverse roles in sperm physiology and/or become a marker for the evaluation of stallion fertility, both of which are worthy of further investigation.     

Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm‐specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca²⁺]_i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT‐PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long‐lasting [Ca²⁺]_i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca²⁺]_i oscillation‐inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.     

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.     

Mechanisms involved in the cytosolic calcium ion elevation induced by activating factors in chicken and rat phagocytes

The aim of the present study was to determine the mechanism of cytosolic calcium ion concentration [Ca²⁺]_i elevation in chicken and rat phagocytes stimulated with phorbol myristate acetate (PMA), leukotriene B₄ (LTB₄), formyl‐methionyl‐leucyl‐phenylananine (fMLP) and Saccaromyces cerevisiae culture supernatant (SCS). Pretreatment with EGTA completely suppressed the PMA‐induced [Ca²⁺]_i elevation in rat and chicken phagocytes, suggesting that all the [Ca²⁺]_i elevation induced in the PMA‐stimulated rat and chicken phagocytes was attributable to the influx of extracellular Ca²⁺. On the other hand, the elevation of LTB₄‐, FMLP‐ and SCS‐induced [Ca²⁺]_i was only partially suppressed by ethyleneglycol‐bis (β‐aminoethyl)‐N,N,N′,N′‐tetraacetic acid ethylene (EGTA) pretreatment of phagocytes. The results indicated that two pathways of [Ca²⁺]_i elevation, recruitment from the intracellular Ca²⁺ store and influx of extracellular Ca²⁺, are involved in the [Ca²⁺]_i elevation of LTB₄‐, fMLP‐ and SCS‐stimulated phagocytes. In fMLP‐stimulated rat neutrophils, [Ca²⁺]_i elevation showed a two‐phase pattern in which the time lag between the first and second phase was approximately 1 min. The EGTA treatment of the fMLP‐stimulated cells induced a reduction of the first phase level and a disappearance of the second phase. The reason for the special influence of EGTA observed in fMLP‐stimulated cells is unknown, but the disappearance of the second phase of the [Ca²⁺]_i may be elicited by the EGTA‐induced decrease of the first phase [Ca²⁺]_i elevation that depends on IP₃ and diacylglycerol induced by fMLP.     

pH-dependent modulation of intracellular free magnesium ions with ion-selective electrodes in papillary muscle of guinea pig

A change in pH can alter the intracellular concentration of electrolytes such as intracellular Ca²⁺ and Na⁺ ([Na⁺]_i) that are important for the cardiac function. For the determination of the role of pH in the cardiac magnesium homeostasis, the intracellular Mg²⁺ concentration ([Mg²⁺]_i), membrane potential and contraction in the papillary muscle of guinea pigs using ion-selective electrodes changing extracellular pH ([pH]_o) or intracellular pH ([pH]_i) were measured in this study. A high CO₂-induced low [pH]_o causes a significant increase in the [Mg²⁺]_i and [Na⁺]_i, which was accompanied by a decrease in the membrane potential and twitch force. The high [pH]_o had the opposite effect. These effects were reversible in both the beating and quiescent muscles. The low [pH]_o-induced increase in [Mg²⁺]_i occurred in the absence of [Mg²⁺]_o. The [Mg²⁺]_i was increased by the low [pH]_i induced by propionate. The [Mg²⁺]_i was increased by the low [pH]_i induced by NH₄Cl-prepulse and decreased by the recovery of [pH]_i induced by the removal of NH₄Cl. These results suggest that the pH can modulate [Mg²⁺]_i with a reverse relationship in heart, probably by affecting the intracellular Mg²⁺ homeostasis, but not by Mg²⁺ transport across the sarcolemma.     

Growth hormone release induced by an amino acid mixture from primary cultured anterior pituitary cells of goats

The effects of amino acids on growth hormone (GH) release and cytosolic calcium concentration ([Ca²⁺]_i) were investigated in caprine anterior pituitary cells cultured for 3 d in Dulbecco modified Eagle medium. The addition of an amino acid mixture consisting of seven nonessential amino acids (NEAA: l-Asp, Gly, l-Ala, l-Ser, l-Pro, l-Asn, and l-Glu; concentration of each 12.5–200 μmol/l) in the medium significantly raised GH release from the cultured cells in a concentration-dependent manner with the maximum release at 200 μmol/l NEAA. Although an addition of l-Asp (0.1–100 μmol/l) caused a significant rise in GH release in a concentration-dependent manner, neither the individual amino acids contained in NEAA except l-Asp nor others (l-Leu, l-Phe, l-Gln, l-Met, and l-Arg) caused a rise in GH release when added alone to the medium. The rise in GH release induced by NEAA (200 μmol/l) and GH-releasing hormone (GHRH, 10 nmol/l) was significantly reduced by the addition of EGTA (l.8 mmol/l) and nifedipine (1 μmol/l) to the medium, respectively. The addition of NEAA (200 μmol/l) caused a rapid and transient [Ca²⁺]_i increase, followed thereafter by a steady increase. The prior addition of nifedipine (1 μmol/l), which itself significantly reduced the basal [Ca²⁺]_i, completely abolished the response induced by NEAA or GHRH. From these findings, we conclude that: 1) NEAA raises GH release and [Ca²⁺]_i in cultured caprine anterior pituitary cells, and 2) Ca²⁺ influx from the medium may be responsible for the cellular action of NEAA.     

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.     

The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

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.     

The concentration of ionized magnesium in serum during the periparturient period of non-paretic dairy cows

Ion-selective electrodes have recently been designed for determining the ionized concentration of magnesium (Mg²⁺) in serum. This development may allow new insights into some metabolic diseases of cattle. For this report, the concentrations of Mg²⁺, total magnesium (Mg_tot), ionized calcium (Ca²⁺), total calcium (Ca_tot), and inorganic phosphate (P_i) were determined in sera from seventeen 3-to 16-year-old Brown Swiss and crossed Simmental/Red Holstein cows during the periparturient period. In each animal, a transient increase of Mg²⁺ and Mg_tot serum concentrations was observed in association with the transient decrease in serum concentrations of Ca²⁺, Ca_tot and P_i after parturition. On average, throughout the study, the serum Mg²⁺ concentrations were 68.5% of those of Mg_tot, whereas the serum Ca²⁺ concentrations were 52% of those of Ca_tot. The possible mechanisms involved in the transient increase of Mg²⁺ and Mg_tot serum concentrations are discussed.Abbreviations Ca²⁺ ionized calcium - Ca_tot total calcium - Mg²⁺ ionized magnesium - Mg_tot total magnesium - P_i inorganic phosphate - PTH parathyroid hormone - PTHrP parathyroid homrone related protein     

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.     

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.     

Increased calcium deposits and decreased Ca-ATPase in erythrocytes of ascitic broiler chickens

The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca²⁺ and Ca²⁺-ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca²⁺-ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca²⁺-ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers.     

Influence of prostaglandin F2α analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro

Although prostaglandin (PG) F_2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF_2α. In the first of two related experiments, the effects of different analogues of PGF_2α (aPGF_2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24 h with naturally-occurring PGF_2α or aPGF_2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca²⁺]_i mobilisation, as well as cell viability and apoptosis were measured.Naturally-occurring PGF_2α and dinoprost stimulated P4 secretion (P < 0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P < 0.001). The greatest effect on [Ca²⁺]_i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P < 0.001).In a second experiment, the influence of naturally-occurring PGF_2α and aPGF_2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF_2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF_2α.     

Endostatin inhibits T-type Ca2+ channel current in guinea pig ventricular myocyte

Endostatin, a fragment of collagen XVIII, is known as an endogenous angiogenesis inhibitor, and its serum concentration increases in various cardiovascular diseases. T-type Ca²⁺ channel, low voltage-activated Ca²⁺ channel, is not expressed in adult ventricular myocytes. Re-expression of T-type Ca²⁺ channels in cardiac myocytes is thought to be involved in the development of cardiac hypertrophy. We examined the effects of endostatin on T-type Ca²⁺ channel current by whole-cell patch clamp technique in freshly isolated adult guinea pig ventricular myocytes, which exceptionally express T-type Ca²⁺ channels. Although endostatin 300 ng/ml had no effect on L-type Ca²⁺ current, it significantly inhibited T-type Ca²⁺ current. These data indicate that endostatin can be an endogenous inhibitor of T-type Ca²⁺ channels in the cardiac myocytes.     

Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine (Gly) can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which Gly affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether Gly could reverse the mitochondrial dysfunction caused by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, which was confirmed by decreased mitochondrial membrane potential (ΔΨm) and the expression of mitochondrial function-related genes PGC-1α, and increased reactiveoxygenspecies (ROS) levelsand the expression of apoptosis-associated genes Bax, Caspase-3, and Cyto C.More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca²⁺]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP₃R1) distribution. Nevertheless, treatment with Gly significantly ameliorated mitochondrial dysfunction, oxidative stress, and apoptosis, and Gly also regulated [Ca²⁺]i levels and IP₃R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes.Taken together, our results indicate that Gly has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.     

Phosphorylation of the MAPK Pathway has an Essential Role in the Acrosome Reaction in Miniature Pig Sperm

In almost all animal species, sperm acrosome reaction (AR) is a crucial step for fertilization. The step is a Ca²⁺‐dependent secretory event that must be completed before fertilization. Many researchers have reported that several chemicals (such as ionomycin, thapsigargin and caffeine) artificially induce this step by increasing [Ca²⁺]_i. However, little information has been known on events that occur following Ca²⁺ induced initiation of the sperm AR. We show here for the first time that phosphorylation of the mitogen‐activated protein kinase (MAPK) pathway is required for the AR in miniature pig sperm. Following caffeine treatment artificially inducing the AR in miniature pig sperm, Raf was phosphorylated and then MAP kinase kinase (MEK) and extracellular‐signal regulated kinase1 (ERK1) were also phosphorylated in a time‐dependent manner. However, the total ERK1 level did not change during the culture. Pre‐treatment of sperm with U0126, a MEK inhibitor, significantly suppressed both the AR and phosphorylation of MEK/ERK1 in a dose‐dependent manner. Additionally, pre‐incubation of the sperm with seminal vesicle (SV) fluid, which is known to contain a decapacitation factor, suppressed both the AR and MEK/ERK1 phosphorylation. These results suggest that phosphorylation of MAPK pathway plays an important role in the AR in miniature pig sperm. Moreover, the SV fluid may have an inhibitory effect on the AR via the suppression of the MAPK pathway.     

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.     

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⁺.