205 CAPACITATION OF STALLION SPERMATOZOA EVALUATED BY FERTILIZATION OF BOVINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 181
Author(s):  
M. R. Hudson ◽  
G. E. Seidel Jr ◽  
E. L. Squires ◽  
B. E. Spizzirri ◽  
D. J. Walker ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Cell Divisions ◽  
Bovine Oocytes

In vitro fertilization in the horse does not work reliably. Several methods of capacitating sperm in other species fail in the horse. The goal of this experiment was to develop a method to capacitate equine spermatozoa using calcium ionophore A23187 or phosphatidylcholine 12 (PC12). We also studied effects of maturing bovine oocytes for 24 or 28 h on fertilizability by capacitated equine sperm, hypothesizing that longer maturation would yield oocytes more easily fertilized by equine spermatozoa. Two sets of bovine oocytes were aspirated from 3 to 8 mm follicles of abattoir ovaries 4 h apart, but fertilized at the same time. On the day of fertilization, semen from 1 of 3 stallions was collected, evaluated, and centrifuged through 33% Percoll to remove seminal plasma. The resultant pellet was extended to 5 × 107 cells mL–1 in M199 containing 0.6% BSA, 2 mm caffeine, and 5 mm CaCl2. Sperm were treated with A23187 (1 or 3 μm) or PC12 (40 or 70 μm) or both A23187 and PC12 (1 μm/40 μm) in 500- μL aliquots. Sperm were incubated at 39°C for 10 min (for A23187 and combination treatments) or 15 min (for PC12 treatments), and then diluted 1:20 for fertilization. Oocytes from each maturation time were fertilized using the same semen preparation for each treatment. Oocytes and sperm were incubated together for 18 h in FCDM in 5% CO2 at 39°C (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596). Presumptive zygotes were cultured for 30 h in CDM-1, vortexed to remove cumulus cells, and evaluated for cleavage. Oocytes were also co-incubated with killed sperm to determine the level of parthenogenesis. Cleaved embryos were stained with orcein to ensure that each cell had a nucleus. Number of cell divisions were recorded as 0 for a 1-cell, 1 for a 2-cell, 1.5 for a 3-cell, etc. More oocytes cleaved after 28 h (18%) than 24 h (14%) maturation (P < 0.01). Sperm of Stallion 1 resulted in higher overall cleavage (24%) than Stallions 2 or 3 (11 and 12%; P < 0.01). Highest cleavage was seen with 28 h maturation and 70 μm PC12 and 3 μm A23187 (27 and 24%, respectively). The most cell divisions were seen with 28 h maturation and 70 μm PC12 (0.48); 28 of the 49 cleaved in this treatment reached ≥4-cell stage. In conclusion, both A23187 and PC12 were able to capacitate equine sperm in a dose-dependent manner as determined from cleavage of bovine oocytes matured for 28 h; maturation for the conventional 24 h was an inferior model for this purpose. Table 1. Mean responses of bovine oocytes fertilized by equine sperm

Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 383 ◽  
Author(s):  
Jin-Tae Chung ◽  
Bruce R. Downey ◽  
Robert F. Casper ◽  
Ri-Cheng Chian
Keyword(s):  
Embryonic Development ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Early Embryonic Development ◽  
Ionophore A23187 ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Treatment Groups ◽  
Bovine Oocytes

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL–1 FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen–thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 M calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v. 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (10.8% v. 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca2+-ionophore. However, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10 000g and stimulation with calcium ionophore A23187 than in the control (18.4% v. 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be ≤7000g to enhance the visibility of nuclear elements for further micromanipulation.

Effect of glucose in the culture medium on development of horse oocytes matured and microfertilized in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1067 ◽  
Author(s):  
T Azuma ◽  
YH Choi ◽  
S Hochi ◽  
N Oguri
Keyword(s):  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Oviduct Fluid ◽  
Effect Of Glucose

The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or 0.1% polyvinyl alcohol (PVA) in 5% O2, 5% CO2 and 90% N2 resulted in very few embryos developing beyond the 8-cell stage. In Experiment 2, the effects of different glucose concentrations (0, 0.5, 5.5 mM) in m-SOF/PVA during Days 1-4 and Days 5-8 of culture were examined. Proportions of oocytes having more than one nucleus ranged from 17.7% to 44.7% among the combinations of glucose concentrations. Supplementation with glucose at 0.5 mM during Days 1-4 followed by 5.5 mM during Days 5-8 resulted in the best embryo development; 12/55 (21.8%) nuclei-positive oocytes developed to the 8-16-cell stage, 11 (20.0%) developed to the 17-50-cell stage, and 5 (9.1%) comprised more than 50 cells and were assumed to be at the morula stage.

139 Effect of protein and calcium ionophore A23187 concentration on hyperactivated motility and acrosome status of stallion sperm

2019 ◽  
Vol 31 (1) ◽  
pp. 195
Author(s):  
I. Ortiz ◽  
H. Resende ◽  
M. Felix ◽  
C. Love ◽  
K. Hinrichs
Keyword(s):  
Repeated Measures ◽  
Calcium Influx ◽  
Semen Analysis ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Computer Assisted ◽  
Ionophore A23187 ◽  
Variable Effect ◽  
Vitro Fertilization

In vitro fertilization does not occur readily in the horse. Fertilization can be achieved using sperm treated with the calcium ionophore A23187 (CaI), but rates are low and variable. In order to fertilize, it is thought that the sperm must show hyperactivated motility and undergo the acrosome reaction. The presence of protein in the media is thought to suppress the effect of CaI, but protein is needed for maintenance of sperm motility. Therefore, the objective of this study was to assess the effect of CaI in the presence or absence of protein on the acrosome and on hyperactivated motility of equine sperm. For this purpose, sperm from 4 stallions were exposed for 10min at 37°C to vehicle or to 1 (C1), 5 (C5) or 10 (C10) μM CaI, with (BSA) or without (N) 7mg mL−1 BSA. The sperm were then washed and incubated at 37°C for 2h. Total and hyperactivated motility were measured by computer-assisted semen analysis. Sperm were considered hyperactivated if curvilinear velocity was &gt;180μm s−1, amplitude of lateral head displacement was &gt;12μm, linearity was &lt;30% and fractal dimension value was &gt;1.3. The percentage of live acrosome-reacted sperm was measured by flow cytometry after staining with propidium iodide and Pisum sativum agglutinin. Data were analysed by repeated-measures 2-way ANOVA. Results were expressed as mean±standard error. Total motility in C5 and C10 treatments was significantly decreased in relation to control (BSA-vehicle) starting at 30min of incubation (35.42±13.57 to 28.20±13.10% v. 71.72±9.21%, respectively; P&lt;0.05). Hyperactivated motility was significantly lower in C10, C5 and N-C1 than in control after 2h of incubation (1.46±0.64v. 3.10±0.58%, respectively). Live acrosome-reacted sperm were significantly higher (P&lt;0.05) for BSA-C5 (14.04±1.99%) and BSA-C10 (14.85±2.52%) than for control (7.50±1.62%) after 2h of incubation. The exposure to sperm of concentrations ≥5μM CaI was associated with loss of motility from 30min of incubation on. However, 2h of incubation after ≥5 μM CaI in the presence of BSA were needed to increase the percentage of live acrosome-reacted sperm. This mismatch between motility and acrosome response helps to clarify the reasons for the variable effect of sperm CaI treatment on equine IVF. Further studies measuring calcium influx and assessing the effect of sperm pre-incubation on CaI response are needed to explore mechanisms for equine in vitro sperm capacitation.

Inhibition of Human Platelet Functions by Verapamil

1981 ◽  
Vol 45 (02) ◽  
pp. 158-161 ◽  
Author(s):  
Y Ikeda ◽  
M Kikuchi ◽  
K Toyama ◽  
K Watanabe ◽  
Y Ando
Keyword(s):  
Platelet Aggregation ◽  
Calcium Ionophore ◽  
Serotonin Release ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dependent Inhibition ◽  
Platelet Functions

SummaryThe effects of verapamil, a coronary vasodilator, on platelet functions were studied.Platelet aggregation induced by ADP, epinephrine or collagen was inhibited by verapamil in vitro. Calcium ionophore A23187-induced platelet aggregation was also inhibited by verapamil in a concentration dependent manner. In washed platelets, verapamil caused a dose-dependent inhibition of serotonin release induced either by thrombin or A23187 in the absence of extracellular calcium. Addition of 1 mM CaCl2 with A23187 or thrombin partially overcame this inhibition. Addition of 1 mM CaCl2 in the absence of verapamil had no effect on thrombin- or A23187-induced secretion. When verapamil was administered to the healthy volunteers at the dosage commonly used, inhibition of platelet aggregation was observed 2 hrs after the drug ingestion. It is of great interest that verapamil potentiated the anti-aggregating activity of prostacyclin in vitro.Our results may suggest a potential role for verapamil in the treatment of thrombotic disorders.

Somatostatin partially reverses desensitization of somatotrophs induced by growth hormone-releasing factor

1987 ◽  
Vol 112 (1) ◽  
pp. 69-76 ◽  
Author(s):  
R. N. Clayton ◽  
L. C. Bailey
Keyword(s):  
Primary Cultures ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Release Mechanism ◽  
Ionophore A23187 ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Response Curves

ABSTRACT The effect of somatostatin on GH-releasing factor (GRF)-induced desensitization of somatotrophs was studied in vitro. Primary cultures of rat anterior pituitary cells pretreated for 4 or 18 h with GRF(1–40) (100 nmol/l) showed a 50% or greater reduction in maximal GH release when rechallenged with 10 nmol GRF/l. Rechallenge GRF dose–response curves were either very flat, making accurate measurement of the dose giving 50% maximum stimulation (ED50) impossible, or the ED50 concentration was increased from 0·3 nmol/l (untreated) to 2 nmol/l (GRF pretreated). Although GRF pretreatment reduced cellular GH content by 40–50%, correction for this did not restore GRF responsiveness measured in terms of maximal GRF-stimulated/unstimulated GH release (maximal/basal ratio), or the GRF ED50 concentration. Maximal/basal GH release per 4 h from GRF-pretreated cells was reduced when cells were rechallenged with forskolin (5 μmol/l) or calcium ionophore (A23187; 10 μmol/l), to the same extent as when rechallenged with 10 nmol GRF/l. Although this might be explained by a reduction in the pool of releasable GH, an alternative explanation is that pretreatment with GRF disrupts the GH release mechanism(s) at a common step(s) beyond cyclic AMP generation and Ca2+ influx. Co-incubation of cells with somatostatin and GRF (100 nmol/l) partially reversed the desensitizing action of GRF during both 4- and 18-h pretreatments in a dose-dependent manner, with 1 μmol somatostatin/l being most effective. Maximal GRF (100 nmol/l)-stimulated/basal GH release was 4·4 ± 1·0 (mean ± s.e.m., n = four experiments), 1·55 ± 0·09 and 2·43 ± 0·1 for control, GRF-pretreated (4 h) and GRF plus somatostatin-pretreated cells respectively. Comparable values for cells pretreated for 18 h were 3·66 ± 0·44 (n = three experiments), 1·78 ± 0·28 and 3·04 ± 0·04 for control, GRF- and GRF plus somatostatin-pretreated cells. Somatostatin reduced the 50% depletion of cellular GH caused by GRF pretreatment to 15–20%, as well as attenuating GH released during the pretreatment period by 40 ± 5% (mean ± s.e.m., n = seven experiments). Somatostatin restored somatotroph sensitivity of GRF-desensitized cells indicating that, in addition to reversing depletion of the releasable pool of GH, the counter-regulatory hormone also prevents disruption of post-receptor cellular biochemical events which remain to be identified. These results add to the list of GRF actions inhibited by somatostatin and suggest a potentially important role for somatostatin in vivo to maintain somatotroph responsiveness to GRF. J. Endocr. (1987) 112, 69–76

Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells

2000 ◽  
Vol 12 (2) ◽  
pp. 15 ◽  
Author(s):  
H. T. Cheong ◽  
K. Ikeda ◽  
M. A. Martinez Diaz ◽  
S. Katagiri ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Calcium Ionophore ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Calcium Ionophore A23187 ◽  
Serum Starvation ◽  
Ionophore A23187 ◽  
Oocyte Collection

This study tested the effects of oocyte collection method, activation protocol and maturational age of recipient oocytes on the in vitro development of nuclear transfer embryos reconstructed with cultured cumulus cells. Cumulus cells synchronized in G0/G1 phase by serum-starvation culture were transferred into enucleated oocytes that were collected by aspiration or dissection method and cultured for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and cycloheximide (CHXM), and cultured for 6 days. Oocyte collection methods, activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44-h-matured recipients. However, the development of embryos reconstituted with 33-h-matured recipients was significantly improved (P<0.05) by activation with the combination of electric pulse and CHXM. The present study shows that reconstituted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage, and that their development can be improved by reconstruction with young oocyte cytoplasts following activation with a combination of electric pulse and CHXM.

Involvement of calmodulin in depolarization-induced release of corticotrophin-releasing hormone-41 from the rat hypothalamus in vitro

1991 ◽  
Vol 7 (1) ◽  
pp. 71-75 ◽  
Author(s):  
S. Tsagarakis ◽  
L. H. Rees ◽  
G. M. Besser ◽  
A. Grossman
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Releasing Hormone ◽  
Rat Hypothalamus ◽  
Maximum Inhibition ◽  
Dose Dependent

ABSTRACT We have employed an acute explant system of the rat hypothalamus in vitro, as previously described, to examine the role of calcium and calmodulin in the release of corticotrophin-releasing hormone-41 (CRH-41). Release of CRH-41, as determined by radioimmunoassay, was stimulated in a dose-dependent manner by the membrane-depolarizing agents KCl and veratridine. Stimulation was also observed with the calcium ionophore A23187. The calcium channel blocker verapamil (1–100 μmol/l) inhibited both KCl-and veratridine-induced release in a dose-dependent manner (maximum inhibition of 75% and 60% respectively), thus providing further evidence that calcium entry is required for secretion of CRH-41 following membrane depolarization. Trifluoperazine (1–100 μmol/1), an inhibitor of calmodulin—calcium interaction, decreased both KCl- and veratridine-evoked CRH-41 secretion in a dose-dependent fashion (maximum inhibition of 50% and 30% respectively). Similarly, phenytoin, a calmodulin-dependent kinase inhibitor, in the concentration range of 1–100 μmol/1, also decreased depolarization-induced CRH-41 release in a dose-dependent manner. The basal release of CRH-41 was unaffected by either treatment. Finally, both calmodulin inhibitors (10 μmol/l) decreased CRH-41 release induced by the calcium ionophore A23187 (10 μmol/l). These data provide evidence for the role of calcium in membrane depolarization-induced stimulus-secretion coupling of rat hypothalamic CRH-41. Furthermore, inhibition of the stimulatory responses by two separate classes of calmodulin inhibitors suggests a role for calmodulin, at least in part, in this process.

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.

Sign in / Sign up

Export Citation Format

Share Document