Puppo A et al. (2008), Alteration of the cortical actin cytoskeleton d...

ECB-ART-40935

PLoS One 2008 Jan 01;310:e3588. doi: 10.1371/journal.pone.0003588.

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Alteration of the cortical actin cytoskeleton deregulates Ca2+ signaling, monospermic fertilization, and sperm entry.

Puppo A , Chun JT , Gragnaniello G , Garante E , Santella L .

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BACKGROUND: When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP(3) receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton. CONCLUSIONS/SIGNIFICANCE: Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.

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Genes referenced: LOC115919910 LOC583082 LOC590297

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	Figure 1. The spatiotemporal relationship among sperm entry, Ca2+ release, and the elevation of the vitelline layer in fertilized starfish eggs.(A) The transmission images of a representative fertilized egg (A. aranciacus) were superimposed with the corresponding fluorograms of the Ca2+ indicator at each time point. The moment of sperm's arrival at the jelly coat was set to t = 0:00 (min:sec). Following the quick cortical flash at 0:26 (arrow), a massive Ca2+ wave initiated from the sperm entry site (0:30) and propagated to the opposite side of the egg. At 3:03 when the Ca2+ wave had already encroached upon the entire cytoplasm, the fertilization envelope began to be elevated (arrow). The fertilization envelope was fully formed only after the Ca2+ wave had traversed the entire cytoplasm at 6:40 (arrow). (B) Detailed views of the sperm entry site (the area marked by a small blue rectangle in panel A) during fertilization. At 2:04 when the vitelline layer is locally elevated, the sperm is still located in the jelly coat (arrow). At 4:00, the sperm head still remains on the outside surface of the egg, but the long acrosomal process (arrow) is inside the egg and connected to a filamentous structure (arrowheads). At 4:42, the sperm head is still visible (arrow), the vitelline layer is further elevated and the focal plasma membrane at the sperm entry site is now being detached from the vitelline layer. At 4:42 and at 6:40, the sperm head (arrow) is still inside the jelly coat. At 7:01, the sperm head is inside the egg cytoplasm (arrow), and the plasma membrane is retracting behind the sperm. The tail is still outside (arrow). At 8:22 and at 9:44, the tail of the sperm finally enters the egg cytoplasm (arrowhead), as the plasma membrane further seals to form a fertilization cone. The motion picture of the entire process is available as a video file (Data S1).
	Figure 2. Heparin induces polyspermy and impedes the propagation of the sperm-induced Ca2+ wave in fertilized starfish eggs.(A) Relative fluorescence pseudo-colored images of the Ca2+ indicator depicting the propagating patterns of Ca2+ waves in the presence or absence of heparin (25 mg/ml, pipette concentration). The moment of the first detectable Ca2+ signal was set to t = 0 in both cases. In heparin-treated eggs, the cortical flash seen in control eggs (arrow) was absent, and polyspermy was evident in all cases (n = 6). The initial Ca2+ spots representing polyspermy were numbered in order of occurrence. (B) Quantification of intracellular Ca2+ levels in fertilized eggs in the control (green curves, n = 4) and heparin-injected (brown curves, n = 6) eggs. (C) The initial phase of the same Ca2+ rise depicted in (B) was plotted in smaller time scale in order to demonstrate the slowed kinetics of the Ca2+ rise in heparin-treated eggs.
	Figure 3. Inhibition of InsP3-dependent Ca2+ release by heparin.Photoactivation of the caged InsP3 (10 µM, pipette concentration) inside A. aranciacus eggs produced massive release of Ca2+ from intracellular stores (green curves, n = 7). In eggs pre-injected with heparin (25 mg/ml, pipette concentration), the Ca2+ response was significantly reduced in its amplitude, but not completely abolished (brown curves, n = 7). The duration of the UV illumination was marked by the blue bar.
	Figure 4. Heparin partially inhibits cADPr-dependent Ca2+ release.(B) Activation of the caged cADPr pre-injected in A. aranciacus eggs results in intracellular release of Ca2+. The altitude of the Ca2+ peak was significantly reduced in heparin-treated eggs compared to the control (n = 7, in each case). (B) Transmission photomicrographs of control and heparin-treated eggs following cADPr uncaging (450 µM, pipette concentration) and subsequent Ca2+ release. The typical elevation of the vitelline layer seen in the control eggs (arrow) was absent in the eggs treated with heparin.
	Figure 5. Heparin alters the cortical actin cytoskeleton and interferes with the formation of the fertilization envelope.(A) The actin cytoskeleton was visualized in living cells by use of fluorescent phalloidin. Mature eggs pre-injected with buffer (control) or heparin (25 mg/ml) were microinjected with fluorescent phalloidin to visualize F-actin. It is evident that heparin induced hyperpolymerization of the cortical actin. (B) The same eggs were fertilized with sperm. In the control egg, the tight cortical actin network dispersed 9 min after the addition of sperm (arrow). The same trend was also evident in one part of the heparin-treated egg (arrow). (C) The control egg displayed normal formation of the fertilization envelope. In the heparin-treated egg, the formation of the fertilization envelope was blocked on the side where the tight cortical actin network did not disperse (arrowhead). (D) In the subplasmalemmal regions of the fertilized eggs visualized by electron microscopy, the images of the cortical granule cores (arrows) are occasionally captured in the perivitelline space of the control eggs. In contrast, heparin-treated eggs exhibited a pile of cortical granules (arrows) in the subplasmalemmal region (E).
	Figure 6. Heparin induces polyspermy and abnormal formation of fertilization cones.Formation of the fertilization cone was monitored in living eggs after sperm addition. (A) F-actin was visualized by microinjection of fluorescent phalloidin. (B) Transmission photomicrographs of the same eggs. Both phalloidin-stained actin networks and the transmission photomicrographs displayed formation of single and round fertilization cone (arrow) in the control egg by 9 min after adding sperm. In contrast, heparin-treated eggs displayed abnormal formation of multiple and piriform-shaped fertilization cones (arrowheads).
	Figure 7. Effects of the actin-polymerizing agent jasplakinolide (JAS) on fertilization.(A) Mature eggs of A. aranciacus were injected with Ca2+ dye and incubated in the presence or absence of JAS (12 µM for 20 min). The moment of the first detectable Ca2+ release was set to t = 0:00 (min:sec). Conspicuous accumulation of Ca2+ dyes was evident in the submembraneous zones of the JAS-incubated eggs (arrow). (B) At 0:07, the control eggs manifested the cortical flash of Ca2+ (arrow), which is absent in the JAS-incubated eggs. Instead, JAS induced polyspermy and produced multiple initiation sites of Ca2+ signals at 0:14 (arrowheads). (C) Quantification of intracellular Ca2+ levels in the control and the JAS-incubated eggs after the addition of sperm. The arrow represents the cortical flash that is absent in the JAS-incubated eggs. (D) The formation of the fertilization envelope seen in the control eggs (arrow) is totally blocked in the JAS-incubated eggs. (E) Comparison of the cortical actin networks in the control and JAS-incubated eggs (before fertilization) using fluorescent phalloidin. In the presence of JAS, starfish eggs displayed remarkable actin hyperpolymerization in the subplasmalemmal region. In contrast, actin fibers in the inner cytoplasm were often reduced by JAS, reflecting the depletion of monomeric actin pool inside the cell [40].
	Figure 8. Heparin blocks sperm entry.The fertilization process in the heparin-injected egg was monitored with a CCD camera, and the key moments were presented by still-shot photomicrographs. The moment of sperm attachment to the egg surface was set to t = 0:00 (min:sec). At 0:41, the sperm was still attached to the jelly coat (arrow). Afterwards, the sperm attempts but fails to penetrate the jelly coat. At 5:20, the vitelline layer is visibly elevated, but the sperm is still completely outside the jelly coat. The formation of the fertilization cone is evident under the elevating membrane (arrow). At 7:52, the vitelline layer is further elevated, but the fertilization cone fails to pull in the sperm head. The fertilization envelope is now being established while the sperm is still outside. The motion picture of the entire process is available as a video file in Data S3.
	Figure 9. Effects of the actin-depolymerizing agent JAS on cADPr-dependent Ca2+ mobilization and the elevation of the vitelline layer.Mature eggs of A. aranciacus loaded with Ca2+ dye and caged cADPr were illuminated with UV to activate the caged second messenger. (A) Relative fluorescence images of the Ca2+ indicator depicting the propagating patterns of Ca2+ waves in the presence or absence of JAS (6 µM). The photoactivation of cADPr initiated Ca2+ release from multiple sites and produced the characteristic cortical Ca2+ signals (arrow) in the control eggs. The strong cortical Ca2+ release seen in the control was absent in the JAS-incubated eggs. (B) Comparison of the intracellular Ca2+ release in the control (green curve) and JAS-incubated eggs after fertilization (brown curve). (C) Photoactivation of cADPr and subsequent release of Ca2+ elevated the vitelline layer in control eggs (arrow). The elevation of the vitelline layer was blocked in the JAS-incubated eggs.
	Figure 10. Effects of the actin depolymerizing agent latruncunlin A (LAT-A) on fertilization.Mature eggs of A. pectinifera were injected with Ca2+ dye and incubated with or without LAT-A (3 µM 30 min) before fertilization. (A) Intracellular Ca2+ release during fertilization. LAT-A slightly lowered the Ca2+ release during fertilization, but the cortical flash (the small initial Ca2+ peak) in the LAT-A-treated eggs (brown curve) was evidently enhanced in comparison with that of the control eggs (green curve). (B) The elevation of the vitelline layer seen in the control eggs (arrow) was completely blocked during the fertilization of the LAT-A treated eggs.

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