Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.

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A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus

Zygote ◽

10.1017/s0967199401001083 ◽

2001 ◽

Vol 9 (1) ◽

pp. 83-88 ◽

Cited By ~ 29

Author(s):

Koji Nakagawa ◽

Shuji Yamano ◽

Hisayo Nakasaka ◽

Kenji Hinokio ◽

Midori Yoshizawa ◽

...

Keyword(s):

Polar Body ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Control Group ◽

Ionophore A23187 ◽

Cleavage Rate ◽

Parthenogenetic Activation ◽

Second Polar Body ◽

Activation Rates ◽

Pronuclear Formation

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 μM A23187 for 5 min were treated with 10 μg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

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Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes

Zygote ◽

10.1017/s096719940000099x ◽

2000 ◽

Vol 8 (3) ◽

pp. 203-208 ◽

Cited By ~ 16

Author(s):

Hisayo Nakasaka ◽

Shuji Yamano ◽

Kenji Hinokio ◽

Koji Nakagawa ◽

Midori Yoshizawa ◽

...

Keyword(s):

Polar Body ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Effective Activation ◽

Control Groups ◽

Mouse Oocytes ◽

Activation Rate ◽

Second Polar Body ◽

And Control

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 μg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.

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Capacity of mouse oocytes to become activated depends on completion of cytoplasmic but not nuclear meiotic maturation

Zygote ◽

10.1017/s0967199400002379 ◽

1995 ◽

Vol 3 (1) ◽

pp. 45-55 ◽

Cited By ~ 8

Author(s):

Joise M.L. McConnell ◽

Liz Campbell ◽

Caroline Vincent

Keyword(s):

Chromosomal Rearrangements ◽

Polar Body ◽

Calcium Ionophore ◽

Germinal Vesicle ◽

Calcium Ionophore A23187 ◽

Meiotic Maturation ◽

Ionophore A23187 ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

Polar Body Extrusion

SummaryThe ability of mouse oocytes to become activated after exposure too the calcium ionophore A23187 has been investigated at different stages of meiotic maturation. The potential to respond to ionophore has been studied in relation to the time since resumption of meiotic maturation, the chromosomal conformation of the DNA within each cell and the protein synthetic profile of the maturing oocyte. Our studies demonstrate that when maturing oocytes from an MF1 strain of mice were treated with A23187 activation occured only in oocytes which had reached second meiotic metaphase (MII). However, development of the ability to respond to ionophore was not dependent on an orderly progression through normal chromosomal rearrangements such as separation at metaphase I (MI) and subsequent polar body extrusion, since there process could be prevented and the capacity to be activated became apparent in such oocytes at a time when control cells had reached MII. These data suggest that the ability to respond to ionophore depends on the development of a cytoplasmic or complex capable of monitoring the time since initiation of germinal vesicle breakdown. Metabolic radiolabelling of oocytes which were able to respond to calcium ionophore, even though they had been prevented from undergoing normal chromosomal rearrangements, showed them to be synthesising a group of proteins known as the 35 kDa complex.

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Induced parthenogenetic activation of oocytes of the marsupial Sminthopsis macroura

Reproduction Fertility and Development ◽

10.1071/rd03054 ◽

2004 ◽

Vol 16 (6) ◽

pp. 599 ◽

Cited By ~ 1

Author(s):

Marek Maleszewski ◽

Lynne Selwood

Keyword(s):

Polar Body ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Protein Synthesis Inhibitor ◽

Ionophore A23187 ◽

Synthesis Inhibitor ◽

Parthenogenetic Activation ◽

First Polar Body ◽

Sminthopsis Macroura ◽

Second Polar Body

Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmental events, has a low success rate and results from the artificial activation of oocytes, which may not include nuclear maturation. In Sminthopsis macroura, 24-h culture of advanced antral follicles in medium containing 10 μg mL−1 porcine pituitary luteinising hormone (LH) yielded 60% of mature polarised oocytes with the first polar body; follicles cultured in medium without LH yielded only immature oocytes. Parthenogenetic activation of follicular, oviducal or uterine oocytes occurred when a two-step protocol was used. Sixty-one oocytes, exposed to 10 μm calcium ionophore A23187 for 10 min followed by 10 μg mL−1 cycloheximide (protein synthesis inhibitor) for 5 h and then cultured for 20–24 h, were scored for signs of activation, namely extrusion of the second polar body and formation of the pronucleus. In each of 43 oocytes (70%), the extruded second polar body was present. Sixteen oocytes were analysed on slides after fixation and staining and, in 13 oocytes (81%) in this group, the female pronucleus was visible. No activation occurred following incubation of oocytes in medium containing Sr2+ for 5 h (n = 14), 8% ethyl alcohol solution for 8 or 12 min (n = 13) or 10 μm calcium ionophore A23187 (n = 13) for 10–20 min followed by culture for 20–24 h.

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Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho

Biochemical Journal ◽

10.1042/bj3270461 ◽

1997 ◽

Vol 327 (2) ◽

pp. 461-472 ◽

Cited By ~ 55

Author(s):

J. Luis GARCÍA ◽

A. Juan ROSADO ◽

Antonio GONZÁLEZ ◽

T. Robert JENSEN

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Inositol Phosphate ◽

Calcium Ionophore ◽

Cytosolic Calcium ◽

Significant Inhibition ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Pancreatic Acini ◽

Pkc Activation

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125FAK and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21rho activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125FAK and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125FAK and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125FAK and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21rho, caused significant inhibition of CCK-8-stimulated p125FAK and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125FAK and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21rho.

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Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽

10.1182/blood.v77.6.1362.1362 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1362-1370 ◽

Cited By ~ 25

Author(s):

JO Hensold ◽

G Dubyak ◽

DE Housman

Keyword(s):

Transcriptional Activation ◽

Calcium Ionophore ◽

Erythroid Differentiation ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Cellular Processes ◽

Cellular Events ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia

Abstract Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

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Rapid ultrastructural changes in the dense tubular system following platelet activation

Blood ◽

10.1182/blood.v80.3.718.718 ◽

1992 ◽

Vol 80 (3) ◽

pp. 718-723 ◽

Cited By ~ 29

Author(s):

L Ebbeling ◽

C Robertson ◽

A McNicol ◽

JM Gerrard

Keyword(s):

Intracellular Calcium ◽

Platelet Activation ◽

Calcium Ionophore ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Ultrastructural Changes ◽

Ionophore A23187 ◽

Tubular System ◽

Protein Kinase C Activators ◽

Morphologic Changes

Abstract The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5′-dimethyl-bis-(0- aminophenoxy)-ethane-N,N,N′,N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

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Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The Journal of Cell Biology ◽

10.1083/jcb.102.4.1459 ◽

1986 ◽

Vol 102 (4) ◽

pp. 1459-1463 ◽

Cited By ~ 73

Author(s):

R I Sha'afi ◽

J Shefcyk ◽

R Yassin ◽

T F Molski ◽

M Volpi ◽

...

Keyword(s):

Intracellular Calcium ◽

Pertussis Toxin ◽

Incubation Medium ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Extracellular Calcium ◽

Intracellular Concentration ◽

The Other ◽

Free Calcium ◽

Ionophore A23187

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.

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The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells

Bioscience Reports ◽

10.1007/bf01121918 ◽

1984 ◽

Vol 4 (7) ◽

pp. 605-611 ◽

Cited By ~ 36

Author(s):

Robert D. Burgoyne

Keyword(s):

Chromaffin Cells ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Catecholamine Release ◽

Intracellular Free Calcium ◽

Free Calcium ◽

Ionophore A23187 ◽

Adrenal Chromaffin Cells ◽

Adrenal Chromaffin ◽

The Relationship

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca2+]i, in bovine adrenal chromaffin cells was determined. At 10−4M carbamylcholine maximal release occurred with an accompanying increase in [Ca2+]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca2+]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca2+]i.

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Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽

10.1182/blood.v77.6.1362.bloodjournal7761362 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1362-1370 ◽

Cited By ~ 1

Author(s):

JO Hensold ◽

G Dubyak ◽

DE Housman

Keyword(s):

Transcriptional Activation ◽

Calcium Ionophore ◽

Erythroid Differentiation ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Cellular Processes ◽

Cellular Events ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

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