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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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Related in: MedlinePlus

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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pone-0003588-g010: 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.

Mentions: Since heparin not only inhibited InsP3 receptors but also induced actin hyperpolymerization (Fig. 5), the cause for the repression of Ca2+ signaling in fertilized eggs pretreated with heparin (Fig. 2) is not straightforward. The finding that heparin, which is not known to interfere with ryanodine receptors, substantially inhibited the Ca2+ release by cADPr (Fig. 4), supported the idea that changes of the actin cytoskeleton may contribute to the Ca2+ release process. To further test this idea, we have used other agents that either polymerize or depolymerize actin filaments. The consequence of the actin hyperpolymerization by JAS has been already mentioned in the context of fertilization (Fig. 7). As shown in Fig. 9A, the cADPr-induced Ca2+ release was also significantly affected by JAS. Liberation of uncaged cADPr in control and JAS-treated eggs gave rise to Ca2+ release from several spots. However, the massive and global release of Ca2+ characteristically seen in the cortex of control eggs (Fig. 9A, arrow) was much compromised in the JAS-treated ones. As a whole, the amplitude of the cADPr-evoked Ca2+ signal was substantially lowered by JAS (Fig. 9B). However, the Ca2+ wave eventually propagated to the cortex; and with this comparably massive Ca2+ signaling, cortical granule exocytosis should have occurred. Nonetheless, the vitelline layer elevation, which results from the cortical granule exocytosis in the control eggs (Fig. 9C, arrow), was totally missing in JAS-treated eggs. These results suggest that the enhancement of F-actin content in the cortex is linked to the inhibition of cortical Ca2+ release and to the blockade of cortical granule exocytosis. Similar experiments with the actin-depolymerizing agent LAT-A led to slightly different effects upon Ca2+ signaling, but had the same effect on vitelline layer elevation seen on the JAS-incubated eggs (Fig. 10A). LAT-A led to diminished Ca2+ signals during the fertilization of A. pectinifera eggs. However, the characteristic cortical flash was significantly increased in LAT-A-treated eggs, suggesting that the decreased cortical layer of polymerized actin [33] was more permissive toward the generation of the cortical flash. On the other hand, the elevation of vitelline layer was totally blocked in LAT-A-treated eggs as well, indicating that not only the hyperpolymerization but also the hypopolymerization of cortical actin had a negative impact on the process of cortical granule exocytosis (Fig. 10B).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003588-g010: 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.
Mentions: Since heparin not only inhibited InsP3 receptors but also induced actin hyperpolymerization (Fig. 5), the cause for the repression of Ca2+ signaling in fertilized eggs pretreated with heparin (Fig. 2) is not straightforward. The finding that heparin, which is not known to interfere with ryanodine receptors, substantially inhibited the Ca2+ release by cADPr (Fig. 4), supported the idea that changes of the actin cytoskeleton may contribute to the Ca2+ release process. To further test this idea, we have used other agents that either polymerize or depolymerize actin filaments. The consequence of the actin hyperpolymerization by JAS has been already mentioned in the context of fertilization (Fig. 7). As shown in Fig. 9A, the cADPr-induced Ca2+ release was also significantly affected by JAS. Liberation of uncaged cADPr in control and JAS-treated eggs gave rise to Ca2+ release from several spots. However, the massive and global release of Ca2+ characteristically seen in the cortex of control eggs (Fig. 9A, arrow) was much compromised in the JAS-treated ones. As a whole, the amplitude of the cADPr-evoked Ca2+ signal was substantially lowered by JAS (Fig. 9B). However, the Ca2+ wave eventually propagated to the cortex; and with this comparably massive Ca2+ signaling, cortical granule exocytosis should have occurred. Nonetheless, the vitelline layer elevation, which results from the cortical granule exocytosis in the control eggs (Fig. 9C, arrow), was totally missing in JAS-treated eggs. These results suggest that the enhancement of F-actin content in the cortex is linked to the inhibition of cortical Ca2+ release and to the blockade of cortical granule exocytosis. Similar experiments with the actin-depolymerizing agent LAT-A led to slightly different effects upon Ca2+ signaling, but had the same effect on vitelline layer elevation seen on the JAS-incubated eggs (Fig. 10A). LAT-A led to diminished Ca2+ signals during the fertilization of A. pectinifera eggs. However, the characteristic cortical flash was significantly increased in LAT-A-treated eggs, suggesting that the decreased cortical layer of polymerized actin [33] was more permissive toward the generation of the cortical flash. On the other hand, the elevation of vitelline layer was totally blocked in LAT-A-treated eggs as well, indicating that not only the hyperpolymerization but also the hypopolymerization of cortical actin had a negative impact on the process of cortical granule exocytosis (Fig. 10B).

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