Vasilev F et al. (2012), Effects of ionomycin on egg activation and earl...

ECB-ART-42480

PLoS One 2012 Jan 01;76:e39231. doi: 10.1371/journal.pone.0039231.

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Effects of ionomycin on egg activation and early development in starfish.

Vasilev F , Chun JT , Gragnaniello G , Garante E , Santella L .

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Ionomycin is a Ca(2+)-selective ionophore that is widely used to increase intracellular Ca(2+) levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs'' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca(2+) levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca(2+) increase. The ionomycin-induced Ca(2+) rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca(2+) response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation.

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Genes referenced: LOC115919910 LOC115925415 LOC583082 LOC590297 stk36

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	Figure 1. Changes of intracellular Ca2+ levels in the starfish oocytes exposed to ionomycin. A. aranciacus oocytes at the GV stage were microinjected with Calcium Green/Rhodamine Red and subsequently exposed to 5 ÂµM ionomycin in artificial seawater in the presence (ASW) or absence (CaFSW) of 10 mM Ca2+. Ca2+ images were then captured with epifluorescence microscopy as described in Materials and Methods. (A) The pseudocolored images of Ca2+ changes within the representative oocytes at several key time points. Indicated by an arrow is the cortical flash. (B) The trajectories of the Ca2+ responses quantified at the entire cytoplasmic field. The Ca2+ responses in ASW and CaFSW are represented in green and brown curves, respectively. To compare the kinetics of the Ca2+ rises in ASW and CaFSW, the moment of the first detectable Ca2+ signal was set to tâ€Š=â€Š0 in panels A and B. (C) The initial response of the oocytes to ionomycin in ASW (green curves) and CaFSW (brown curves). To better illustrate the difference in the time lag before the first detectable Ca2+ rise, the moment of the ionomycin addition was set to tâ€Š=â€Š0 in this panel.
	Figure 2. Morphological changes in the cortex of the starfish oocytes exposed to ionomycin. A. aranciacus oocytes at the GV stage were fixed in glutaraldehyde after 5 min incubation with 5 ÂµM ionomycin in natural seawater. (A) Bright field view in the light microscope. GVâ€Š=â€Šgerminal vesicle. Scale barâ€Š=â€Š50 Âµm. (B) The magnified views of the dot-lined rectangular areas in panel A. The same large vesicles in panel A were marked with yellow arrowheads. Note that cortical granules that appear as dark vesicles sized about 1 Âµm (arrow) had largely disappeared in the oocytes briefly exposed to ionomycin. Scale barâ€Š=â€Š10 Âµm. (C) TEM image of the same batch of oocytes incubated in the absence (left) or present of 5 ÂµM ionomycin for 5 min. Blue arrowheads indicate microvilli in cross-section. Red arrows indicate the white vesicles engulfing electron-dense cortical granules. Blue arrows, white vesicles at fusion; Scale barâ€Š=â€Š10 Âµm.
	Figure 3. Ionomycin induces rapid rearrangement of the actin cytoskeleton.(A) A living oocyte (A. aranciacus) microinjected with Alexa Fluor 488-conjugated phalloidin was exposed to 5 ÂµM ionomycin and monitored under the confocal microscope. Note the continuous layer of the subplasmalemmal actin network delineating the plasma membrane before the ionomycin treatment (arrow, tâ€Š=â€Š0) had mostly disappeared within 5 min in the same oocytes. In contrast, the actin filaments in the inner cytoplasm formed bundles and became much thicker and longer. (B) After the brief exposure (5 min) to 5 ÂµM ionomycin, the oocytes were switched to normal seawater without ionomycin and induced to undergo meiotic maturation in the presence of 10 ÂµM 1-MA for 1 hour. The orderly arranged actin filaments seen in the control eggs (arrowheads) are mostly lost in the eggs briefly exposed to ionomycin at the GV stage. Instead, a thick layer of actin fibers surrounded the big fused white vesicles (arrows).
	Figure 4. Disruption of cortical granules and microvilli by the brief exposure to ionomycin leads to depletion of the ionomycin-sensitive Ca2+ stores. A. aranciacus oocytes at the GV stage were exposed to 5 ÂµM ionomycin for 3 min before switched to the media containing 1-MA. (A) After 1 h incubation, the mature eggs were fixed with glutaraldehyde and analyzed by TEM. Blue arrows indicate the remnant of the cortical granules that were extruded in the perivitelline space. Red arrows indicate fragments of cortical granules being engulfed by white vesicles. Scale barâ€Š=â€Š10 Âµm. (B) The same batch of oocytes were exposed to 5 ÂµM ionomycin for 3 min and switched to the fresh media containing 1-MA. After GV breakdown, the mature eggs were microinjected with Calcium Green/Rhodamine Red and subsequently re-exposed to 5 ÂµM ionomycin (tâ€Š=â€Š0) to monitor the Ca2+ response. The trajectory of intracellular Ca2+ levels in the eggs with or without (control) ionomycin pretreatment were depicted in brown and green curves, respectively.
	Figure 5. The mature eggs pretreated with ionomycin at the GV stage respond to the second dose of ionomycin or A23187 with no intracellular Ca2+ increase. P. miniata oocytes at the GV stage were briefly exposed to 5 ÂµM ionomycin and switched to the normal seawater containing 10 ÂµM 1-MA for 1 h and subsequently challenged with the second dose of 5 ÂµM ionomycin (A) or 40 ÂµM A23187 (B). In both cases, the green curves depict the Ca2+ response in the control eggs, and the brown ones the response of the eggs that had been briefly exposed to 5 ÂµM ionomycin at the GV stage.
	Figure 6. Ionomycin-exposed eggs with cortical granule disruption still respond to InsP3 with an intracellular Ca2+ release, but to a reduced extent. A. aranciacus oocytes were exposed to 5 ÂµM ionomycin for 5 min at the GV stage and microinjected with caged InsP3 and Calcium Green. The oocytes were matured in the fresh seawater containing 10 ÂµM 1-MA and then irradiated with UV to photoactivate the Ca2+-mobilizing second messenger. (A) Results of one of the three independent experiments showing the trajectories of the quantified Ca2+ responses at the entire cytoplasmic field. Ca2+ responses in the control eggs and the eggs briefly exposed to 5 ÂµM ionomycin at the GV stage were shown in green and brown curves, respectively. Violet line indicates the duration of UV irradiation. (B) Summary of the data pooled from three independent batches of experiments comprising 3 or 4 microinjected eggs with (brown bars, nâ€Š=â€Š10) or without (control, green bars; nâ€Š=â€Š9) ionomycin pretreatment at the GV stage. The average amplitude (mean Â± standard deviation, left histogram) and the time interval between the onset and the peak of the Ca2+ signals (right histogram) were depicted separately. Asterisk indicates a significant difference between the control and the ionomycin-pretreated eggs (p<0.0001). (C) Despite the substantial amount of Ca2+ released, the ionomycin-pretreated eggs did not undergo elevation of the vitelline layer in all cases.
	Figure 7. Fertilization of the ionomycin-pretreated eggs with altered cortical structure. A. aranciacus oocytes were briefly exposed to ionomycin (5 ÂµM for 5 min) at the GV stage and subsequently incubated in fresh seawater containing 10 ÂµM 1-MA. The mature eggs were then inseminated. (A) Results of one of the five independent experiments. The trajectories of the quantified Ca2+ responses at the entire cytoplasmic field. Ca2+ responses in the control eggs and the eggs briefly exposed to 5 ÂµM ionomycin at the GV stage were shown in green and brown curves, respectively. To illustrate the difference in the latent period before the Ca2+ response, the moment of the fertilizing sperm addition was set to tâ€Š=â€Š0. Asterisks indicate the Ca2+ peaks of the eggs that required a second addition of sperm (5 min after the first insemination). (Bâ€“F) Summary of the data pooled from five independent batches of experiments comprising 4 to 8 eggs with (brown bars, nâ€Š=â€Š20) or without (control, green bars; nâ€Š=â€Š23) ionomycin pretreatment at the GV stage. The average amplitude (mean Â± standard deviation) of the Ca2+ peaks and the time interval between the onset and the peak of the signals were plotted in panels B and C, respectively. (D) Pseudocolor images of the representative cortical flashes in the control and the ionomycin-pretreated eggs (arrow) from the same batch of experiment. Ca2+ images were captured with epifluorescence microscopy as described in the Materials and Methods. (E) Frequency of the detectable cortical flashes in the same five independent experiments. (F) Comparison of the amplitude of the cortical flashes. Data were normalized in reference to the average value of the control eggs in each batch of experiment.
	Figure 8. Microinjected ionomycin does not induce Ca2+ increase inside the starfish eggs. P. miniata oocytes were microinjected with Calcium Green and induced to mature in 10 ÂµM 1-MA for 1 h. Under the CCD camera, the mature eggs were microinjected with InsP3 (without caging, 5 ÂµM in pipette tip), ionomycin (50 ÂµM), or the injection buffer only. Results of one of the three independent experiments are shown. (A) Transmission views of the eggs 10 min after microinjection. (B) Quantified Ca2+ signals for InsP3 (blue curve), injection buffer (green), and ionomycin (brown).
	Figure 9. Fertilization and the early development of the ionomycin-pretreated eggs.(A) Astropecten aranciacus oocytes were pretreated with 5 ÂµM ionomycin at the GV stage and matured with 1-MA for 1 h. Subsequently, eggs with or without (control) ionomycin pretreatment were fertilized with Hoechst 33342-stained sperm (see Materials and Methods). After 20 min, the number of the internalized sperm in each egg was counted, and the frequencies of monospermy (gray bars), polyspermy (black bars, sperm count >2), or the case with no evident sperm entry (white bar) were calculated in four independent experiments. (B) Developmental progress of the representative control and the ionomycin-pretreated eggs that clearly established monospermic sperm entry (arrows). (C) Summary of the fertilization envelope (FE) formation and the rate of abnormal development in the control and the ionomycin-pretreated eggs that established monospermic zygotes.
	Figure 10. Ionomycin pretreatment disrupts the functionality of the cortical actin cytoskeleton. P. miniata oocytes were exposed to ionomycin (5 ÂµM for 8 min) at the GV stage and subsequently incubated in fresh seawater containing 10 ÂµM 1-MA. The mature eggs were then microinjected with Alexa Fluor 488-phalloidin and inseminated to monitor with confocal microscopy the real-time changes of the actin cytoskeleton. In the control eggs, the orderly arranged subplasmalemmal actin filaments migrated centripetally (arrow), which was synchronized with the elevation of the fertilization envelope. In contrast, mature eggs previously exposed to 5 ÂµM at the GV stage failed to show such migration. At the right side of each panel, the fluorescence image of F-actin in confocal microscopy was merged with the transmission view of the same specimen. Images of the eggs before and after fertilization (13 min post-insemination) were taken from the same individual eggs.
	Figure 11. Deleterious effects of ionomycin on development.Mature eggs of Astropecten aranciacus were fertilized with Hoechst 33342-prestained sperm. After 20 min, zygotes displaying clear signs of monospermy and fully elevated fertilization envelope were exposed either to 5 ÂµM ionomycin or to 0.1% DMSO (control, vehicle) for 10 min and further incubated in seawater to monitor the developmental progress. (A) Representative photomicrographs of the early embryos developing from the control and the monospermic zygotes exposed to ionomycin. For the latter, only the normally growing ones at 4 h and 3 d after fertilization were marked with arrows. (B) Frequency of normal development in the control and the ionomycin-exposed zygotes. Data were pooled from three independent batches of experiments comprising 8 to 10 monospermic zygotes with (brown bars) or without (control, green bars) the 10 min ionomycin treatment after fertilization.

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