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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-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].
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pone-0003588-g007: 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].

Mentions: Since heparin induced polyspermy, we examined how it affected the formation of fertilization cones in starfish eggs. In the heparin-treated eggs loaded with phalloidin, the average number of fertilization cones was 6.4 per cell (n = 10), while it was 1.6 (n = 10) in the control eggs. Although the fertilization cones in both cases were rich in F-actin (Fig. 6A, arrow and arrowheads), the heparin-treated eggs exhibited a substantial concentration of actin fibers in the cortical domain underneath the fertilization cones (Fig. 6A). Furthermore, the conical shape of the fertilization cones in heparin-treated eggs (Fig. 6, arrowheads) was at variance with that of control eggs, which resembled a round bleb (Fig. 6, arrows). These fundamental differences in number and shape of fertilization cones were not due to the presence of phalloidin, as similar transmission light microscopy experiments without phalloidin produced the same results (not shown). A plausible concern at this point regards the way heparin induced polyspermy and abnormal formation of the fertilization cones, i.e., whether it did so by inhibiting the InsP3R, or by alterating the actin cytoskeleton at the subplasmalemmal region of the egg [33]. To test whether polyspermy and the changes of Ca2+ signaling were linked to the alteration of the cortical actin cytoskeleton, we examined the effect of jasplakinolide (JAS), a well characterized agent that stimulates cortical actin polymerization [40]. As was the case for heparin, fertilization of the starfish eggs pre-incubated with JAS produced polyspermy, as indirectly shown by the presence of multiple initiation sites of Ca2+ waves that represent loci of sperm interaction (Fig. 7B, arrowheads). The most evident effect of JAS was the elimination of the cortical flash of control eggs (Fig. 7B, arrow at 0:07). This effect is evident in the quantified Ca2+ curves, where the small peak at the very beginning of the fertilization-induced Ca2+ rise was blocked by JAS (Fig. 7C, arrow). On the other hand, the overall pattern of the Ca2+ waves at later stages of fertilization was virtually the same as in the control egg. However, despite the massive release of Ca2+ that reached the cortex of the cell, JAS completely blocked the vitelline layer elevation seen in the control egg (Fig. 7D, arrow). An additional difference between the JAS-incubated and the heparin-treated eggs is that the former produced no fertilization cone despite the initial polyspermic interactions and the multiple foci of local Ca2+ release (Fig. 7B), as the formation of the fertilization cone is associated with vitelline layer elevation (Fig. 1B). F-actin visualized by phalloidin became dramatically concentrated, corroborating the suggestion that all these changes on the egg surface are linked to the alteration of the cortical actin cytoskeleton in the subplasmalemmal region of the JAS-treated eggs (Fig. 7E).


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-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].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003588-g007: 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].
Mentions: Since heparin induced polyspermy, we examined how it affected the formation of fertilization cones in starfish eggs. In the heparin-treated eggs loaded with phalloidin, the average number of fertilization cones was 6.4 per cell (n = 10), while it was 1.6 (n = 10) in the control eggs. Although the fertilization cones in both cases were rich in F-actin (Fig. 6A, arrow and arrowheads), the heparin-treated eggs exhibited a substantial concentration of actin fibers in the cortical domain underneath the fertilization cones (Fig. 6A). Furthermore, the conical shape of the fertilization cones in heparin-treated eggs (Fig. 6, arrowheads) was at variance with that of control eggs, which resembled a round bleb (Fig. 6, arrows). These fundamental differences in number and shape of fertilization cones were not due to the presence of phalloidin, as similar transmission light microscopy experiments without phalloidin produced the same results (not shown). A plausible concern at this point regards the way heparin induced polyspermy and abnormal formation of the fertilization cones, i.e., whether it did so by inhibiting the InsP3R, or by alterating the actin cytoskeleton at the subplasmalemmal region of the egg [33]. To test whether polyspermy and the changes of Ca2+ signaling were linked to the alteration of the cortical actin cytoskeleton, we examined the effect of jasplakinolide (JAS), a well characterized agent that stimulates cortical actin polymerization [40]. As was the case for heparin, fertilization of the starfish eggs pre-incubated with JAS produced polyspermy, as indirectly shown by the presence of multiple initiation sites of Ca2+ waves that represent loci of sperm interaction (Fig. 7B, arrowheads). The most evident effect of JAS was the elimination of the cortical flash of control eggs (Fig. 7B, arrow at 0:07). This effect is evident in the quantified Ca2+ curves, where the small peak at the very beginning of the fertilization-induced Ca2+ rise was blocked by JAS (Fig. 7C, arrow). On the other hand, the overall pattern of the Ca2+ waves at later stages of fertilization was virtually the same as in the control egg. However, despite the massive release of Ca2+ that reached the cortex of the cell, JAS completely blocked the vitelline layer elevation seen in the control egg (Fig. 7D, arrow). An additional difference between the JAS-incubated and the heparin-treated eggs is that the former produced no fertilization cone despite the initial polyspermic interactions and the multiple foci of local Ca2+ release (Fig. 7B), as the formation of the fertilization cone is associated with vitelline layer elevation (Fig. 1B). F-actin visualized by phalloidin became dramatically concentrated, corroborating the suggestion that all these changes on the egg surface are linked to the alteration of the cortical actin cytoskeleton in the subplasmalemmal region of the JAS-treated eggs (Fig. 7E).

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