Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle

1999 ◽  
Vol 11 (1) ◽  
pp. 59 ◽  
Author(s):  
Hong Wei ◽  
Yutaka Fukui
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Formation Rate ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Significant Difference ◽  
Bull Sperm ◽  
Chemical Pretreatments ◽  
Pronuclear Formation

This study investigated the effects of the bull, sperm type (dead, immotile or motile) and sperm pretreatment (i.e. mechanical (tail-cutting or tail-scoring) or chemical (heparin, heparin + caffeine, calcium ionophore A23187 or dithiothreitol)) on male pronuclear formation after intracytoplasmic sperm injection (ICSI) in cattle. Three experiments were conducted. In Experiment 1, spermatozoa from three bulls (A, B and C) were used for both ICSI and in vitro fertilization (IVF). The results were that sperm from bull B yielded a higher penetration/male pronuclear formation rate than that of bull C when used for IVF (89.6% v. 25.6%, P<0.01). However, when injected into oocytes by ICSI, sperm from bull C had a higher male pronuclear formation rate than that of bull B (34.6% v. 16.1%, P<0.05). The effects of sperm type and mechanical pretreatment were examined in Experiment 2. No significant difference was found in the male pronuclear formation rate when the three types of sperm were injected into oocytes. Tail-scored sperm achieved a higher male pronuclear rate than that of non-mechanically treated ones (38.2% v. 13.2%, P<0.005). In Experiment 3, chemical pretreatments were tested and compared. Higher male pronuclear rates, compared with the control, were obtained when sperm were pretreated with heparin + caffeine, calcium ionophore A23187 and dithiothreitol (48.2%, 62.5% and 64.5% v. 25.0%, P<0.05, 0.005 and 0.005, respectively). These results indicate that (1) there is a bull variation in male pronuclear formation with ICSI, and the male pronuclear rate by ICSI is not coincident with the results by IVF, (2) immobilization of a spermatozoon by tail-scoring before ICSI can improve the formation of the male pronucleus, and (3) an appropriate chemical pretreatment of spermatozoa is necessary to achieve a higher rate of male pronuclear formation.

376 FAILURE OF SPERM DECONDENSATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN SWAMP BUFFALO (BUBALUS BUBALIS)

2010 ◽  
Vol 22 (1) ◽  
pp. 344
Author(s):  
V. Chankitisakul ◽  
A. Tharasanit ◽  
K. Thaseephoo ◽  
M. Techakumphu
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Chemical Activation ◽  
Formation Rate ◽  
Calcium Ionophore ◽  
Developmental Competence ◽  
Ionophore A23187 ◽  
Blastocyst Formation ◽  
Swamp Buffalo ◽  
Pronuclear Formation

Intracytoplasmic sperm injection (ICSI) has been intensively used to examine the early events of gamete activation, but few studies have been reported for swamp buffalo. The first objective (Exp. 1) was to compare the developmental competence of oocytes after ICSI using either live or dead frozen-thawed spermatozoa. Matured oocytes were fertilized by ICSI using live (n = 148) or dead (n = 151) spermatozoa, followed by chemical activation using calcium ionophore (A23187) and cyclohexamide (CHX) in SOF medium. In vitro fertilization (n = 149) served as thecontrol. Cleavage rate was recorded on Day 2 and blastocyst formation rate was evaluated on Day 7. The second objective (Exp. 2) was to examine the effects of ICSI and activation regime on the decondensation of buffalo spermatozoa. A total of 148 matured oocytes were subjected to ICSI. The sperm-injected oocytes (n = 87) were then activated using the activation protocol as described in Exp. 1. The ICSI oocytes without activation (n = 61) and sham-injected oocytes with activation (n = 35) were used as controls. Nuclear changes of presumptive zygotes were mor- phologically evaluated for pronuclear formation using 4′,6-diamidino-2-phenylindole procedure and epifluorescent microscopy at 18 h post-ICSI. Statistical differences were determined among the groups using chi-square test. In Exp. 1, the results showed that the percentages of cleavage and blastocyst formation rate were 79.7, 77.0, and 41.6% and 33.8, 30.5, and 14.8% in the live sperm, dead sperm, and IVF groups, respectively. Embryo development rates did not significantly differ between ICSI groups; however, these rates were significantly higher than in the IVF group (P < 0.05). In Exp. 2, the pronuclear formation rate was significantly higher in the ICSI with chemical (70.1%) and sham injection with chemical (60.6%) groups than in the ICSI without chemical group (3.2%; P < 0.01). However, most of the presumptive zygotes with pronuclear formation from the ICSI with chemical activation group showed only intact sperm heads instead of the full male pronuclear formation. Our study suggests that the chemical activation directly affected the female pronuclear formation and embryo development but that it was not associated with the male pronuclear formation. It is postulated that ICSI oocytes that developed to cleavage and blastocyst stages underwent parthenogenesis after chemical activation. This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets

2007 ◽  
Vol 97 (03) ◽  
pp. 425-434 ◽  
Author(s):  
Dmitry Kireev ◽  
Nadezhda Popenko ◽  
Aleksei Pichugin ◽  
Mikhail Panteleev ◽  
Olga Krymskaya ◽  
...  
Keyword(s):  
Blood Platelets ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
In Vitro Models ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor X ◽  
Activated Platelets

SummaryPlatelet microparticles (PMPs) are small vesicles released from blood platelets upon activation. The procoagulant activity of PMPs has been previously mainly characterized by theirability to bind coagulation factors VIII and Va in reconstructed systems. It can be supposed that PMPs can contribute to the development of thrombotic complications in the pathologic states associated with the increase of their blood concentration. In this study we compared procoagulant properties of calcium ionophore A23187-activated platelets and PMPs using several in-vitro models of hemostasis. Surface densities of phosphatidylserine, CD61, CD62P and factor X bound per surface area unit were determined by flow cytometry. They were 2.7-, 8.4-, 4.3-, and 13-fold higher for PMPs than for activated platelets, respectively. Spatial clot growth rate (Vclot) in the reaction-diffus ion experimental model and endogenous thrombin potential (ETP) were determined in plasma, which was depleted of phospholipid cell surfaces by ultra-centrifugation and supplemented with activated platelets or PMPs at different concentrations. Both Vcllot and ETP rapidly increased with the increase of PMP or platelet concentration until saturation was reached. The plateau values of Vclot and ETP for activated platelets and PMPs were similar. In both assays, the procoagulant activity of one PMP was almost equal to that of one activated platelet despite at least two-orders-of-magnitude difference in their surface areas. This suggests that the PMP surface is approximately 50- to 100-fold more procoagulant than the surface of activated platelets.

scholarly journals MiR-302e attenuates allergic inflammation in vitro model by targeting RelA

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Lifeng Xiao ◽  
Li Jiang ◽  
Qi Hu ◽  
Yuru Li
Keyword(s):  
Inflammatory Cytokines ◽  
Luciferase Activity ◽  
Allergic Inflammation ◽  
Calcium Ionophore ◽  
Allergic Diseases ◽  
Calcium Ionophore A23187 ◽  
Human Mast Cell ◽  
Ionophore A23187 ◽  
Down Regulation

Allergic inflammation is the foundation of allergic rhinitis and asthma. Although microRNAs are implicated in the pathogenesis of various diseases, information regarding the functional role of microRNAs in allergic diseases is limited. Herein, we reported that microRNA-302e (miR-302e) serves as an important regulator of allergic inflammation in human mast cell line, HMC-1 cells. Our results showed that miR-302e is the dominant member of miR-302 family expressed in HMC-1 cells. Moreover, the expression of miR-302e was significantly decreased in response to phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 or ovalbumin (OVA) stimulation. Overexpression of miR-302e blocked PMA/A23187 or OVA induced the increase in inflammatory cytokines levels, such as IL-1β, IL-6, tumor necrosis factor (TNF)-α and thymic stromal lymphopoietin, while miR-302 inhibition further promoted the release of these cytokines. Mechanistically, we found that miR-302e is a novel miRNA that targets RelA, a gene known to be involved in regulating inflammation, through binding to the 3′-UTR of RelA mRNA. Ectopic miR-302e remarkably suppressed the luciferase activity and expression of RelA, whereas down-regulation of miR-302e increased RelA luciferase activity and expression. Pharmacological inhibition of NF-κB reversed the augmented effect of miR-302e down-regulation on inflammatory cytokines level. Taken together, the present study demonstrates miR-302e limits allergic inflammation through inhibition of NF-κB activation, suggesting miR-302e may play an anti-inflammatory role in allergic diseases and function as a novel therapeutic target for the treatment of these diseases.

scholarly journals Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Atsushi Enomoto ◽  
Takemichi Fukasawa ◽  
Hiroki Tsumoto ◽  
Masataka Karube ◽  
Keiichi Nakagawa ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Terminal Region ◽  
Calpain Inhibitor ◽  
Ionophore A23187 ◽  
Serine Threonine Kinase ◽  
Defective Mutant ◽  
Cleavage Assay

Abstract Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

scholarly journals Tulathromycin Exerts Proresolving Effects in Bovine Neutrophils by Inhibiting Phospholipases and Altering Leukotriene B4, Prostaglandin E2, and Lipoxin A4Production

2014 ◽  
Vol 58 (8) ◽  
pp. 4298-4307 ◽  
Author(s):  
Carrie D. Fischer ◽  
Stephanie C. Duquette ◽  
Bernard S. Renaux ◽  
Troy D. Feener ◽  
Douglas W. Morck ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Lipid Signaling ◽  
Prostaglandin E ◽  
Ionophore A23187 ◽  
Proinflammatory Mediators ◽  
Bovine Neutrophils

ABSTRACTThe accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4(LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4[LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytesin vitroand inMannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammationin vivoand characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4and prostaglandin E2(PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2(PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

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