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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 - PLoS ONE (2008)

Bottom Line: We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization.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.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.

View Article: PubMed Central - PubMed

Affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy.

ABSTRACT

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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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).
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pone-0003588-g005: 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).

Mentions: We have recently demonstrated that heparin hyperpolymerizes cortical actin in premeiotic oocytes of starfish [33]. We have now found that heparin dramatically increased actin polymerization in the subplasmalemmal region of the postmeiotic eggs as well. Mature eggs of A. aranciacus microinjected with heparin displayed much denser phalloidin labeling in the cortical actin layer (Fig. 5A). This remarkable enhancement of actin polymerization in the cortex is not due to a potential secondary effect of phalloidin itself, which has been known to stabilize F-actin [39]. Glutaraldehyde fixation of the heparin-treated eggs before visualizing actin with phalloidin also exhibited heparin-specific hyperpolymerization of cortical actin (Data S2). The intricate network of actin filaments in the control cortex dispersed soon after fertilization (Fig. 5B). This trend of F-actin to become reorganized was strong enough to be evident even in the major part of the heparin-treated eggs after fertilization (Fig. 5B, arrow). However, the heparin-induced hyperpolymerization of actin prevented the centripetal movement of the actin fibers to the other part of the egg (Fig. 5B, arrowhead). The sperm interaction often failed to elevate the vitelline layer on the entire surface of the heparin-treated eggs, resulting in a partial and polarized formation of the fertilization envelope (Fig. 5C; 6 out of 10 eggs). The suggestion that reorganization of the cortical actin cytoskeleton facilitated the exocytosis of cortical granules was reinforced by the finding that the fertilization of heparin-treated eggs produced elevation of the vitelline layer only in the area from which the cortical actin network had dispersed (Fig. 5B and C, arrows). The regional blockade of the cortical granule exocytosis by heparin was also visualized by electron microscopy. Whereas the control egg exported the dark shaded cortical granules from the cytoplasm (Fig. 5D), the heparin-treated egg conspicuously accumulated them in the subplasmalemmal region adjacent to the partially elevated fertilization envelope (Fig. 5E).


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

Puppo A, Chun JT, Gragnaniello G, Garante E, Santella L - PLoS ONE (2008)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570615&req=5

pone-0003588-g005: 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).
Mentions: We have recently demonstrated that heparin hyperpolymerizes cortical actin in premeiotic oocytes of starfish [33]. We have now found that heparin dramatically increased actin polymerization in the subplasmalemmal region of the postmeiotic eggs as well. Mature eggs of A. aranciacus microinjected with heparin displayed much denser phalloidin labeling in the cortical actin layer (Fig. 5A). This remarkable enhancement of actin polymerization in the cortex is not due to a potential secondary effect of phalloidin itself, which has been known to stabilize F-actin [39]. Glutaraldehyde fixation of the heparin-treated eggs before visualizing actin with phalloidin also exhibited heparin-specific hyperpolymerization of cortical actin (Data S2). The intricate network of actin filaments in the control cortex dispersed soon after fertilization (Fig. 5B). This trend of F-actin to become reorganized was strong enough to be evident even in the major part of the heparin-treated eggs after fertilization (Fig. 5B, arrow). However, the heparin-induced hyperpolymerization of actin prevented the centripetal movement of the actin fibers to the other part of the egg (Fig. 5B, arrowhead). The sperm interaction often failed to elevate the vitelline layer on the entire surface of the heparin-treated eggs, resulting in a partial and polarized formation of the fertilization envelope (Fig. 5C; 6 out of 10 eggs). The suggestion that reorganization of the cortical actin cytoskeleton facilitated the exocytosis of cortical granules was reinforced by the finding that the fertilization of heparin-treated eggs produced elevation of the vitelline layer only in the area from which the cortical actin network had dispersed (Fig. 5B and C, arrows). The regional blockade of the cortical granule exocytosis by heparin was also visualized by electron microscopy. Whereas the control egg exported the dark shaded cortical granules from the cytoplasm (Fig. 5D), the heparin-treated egg conspicuously accumulated them in the subplasmalemmal region adjacent to the partially elevated fertilization envelope (Fig. 5E).

Bottom Line: We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization.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.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.

View Article: PubMed Central - PubMed

Affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus