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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 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.
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pone-0003588-g002: 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.

Mentions: Heparin has been used extensively as an antagonist of InsP3Rs. As has been reported for Xenopus and sea urchin eggs [24], [25], the microinjection of heparin into starfish eggs also increased the occurrence of polyspermy. In heparin-treated eggs, we found that Ca2+ waves evidently initiated at multiple sperm-egg interaction sites (Fig. 2A, lower panel). The average number of detectable initial Ca2+ spots in heparin-treated eggs was 4.8 after fertilization, as opposed to 1.2 in the control eggs (n = 5, each case). In addition, the characteristic cortical flash seen in the control egg was abolished in the heparin-treated eggs. The amplitude and kinetics of the intracellular Ca2+ rise were also significantly reduced (Fig. 2B). The Ca2+ transients peaked at 0.55±0.12 arbitrary units within 272 sec in heparin-treated eggs, as opposed to 0.83±0.03 arbitrary units in 70 sec in the monospermic control eggs. The delayed kinetics and lowered amplitude of the Ca2+ peak in heparin-treated eggs are not due to polyspermy itself, because occasional polyspermy occurring in control eggs displayed an even faster Ca2+ rise (not shown). Considering that heparin is an inhibitor of InsP3 receptors, the inhibition of Ca2+ propagation was not surprising. The treatment with heparin indeed inhibited the Ca2+-releasing activity of injected InsP3. The average amplitude of the Ca2+ peak seen in control eggs (0.55±0.04 arbitrary units) was strongly reduced (0.25±0.05), but not completely abolished (Fig. 3). The significance of this observation will be discussed in more detail later on (see Discussion).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003588-g002: 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.
Mentions: Heparin has been used extensively as an antagonist of InsP3Rs. As has been reported for Xenopus and sea urchin eggs [24], [25], the microinjection of heparin into starfish eggs also increased the occurrence of polyspermy. In heparin-treated eggs, we found that Ca2+ waves evidently initiated at multiple sperm-egg interaction sites (Fig. 2A, lower panel). The average number of detectable initial Ca2+ spots in heparin-treated eggs was 4.8 after fertilization, as opposed to 1.2 in the control eggs (n = 5, each case). In addition, the characteristic cortical flash seen in the control egg was abolished in the heparin-treated eggs. The amplitude and kinetics of the intracellular Ca2+ rise were also significantly reduced (Fig. 2B). The Ca2+ transients peaked at 0.55±0.12 arbitrary units within 272 sec in heparin-treated eggs, as opposed to 0.83±0.03 arbitrary units in 70 sec in the monospermic control eggs. The delayed kinetics and lowered amplitude of the Ca2+ peak in heparin-treated eggs are not due to polyspermy itself, because occasional polyspermy occurring in control eggs displayed an even faster Ca2+ rise (not shown). Considering that heparin is an inhibitor of InsP3 receptors, the inhibition of Ca2+ propagation was not surprising. The treatment with heparin indeed inhibited the Ca2+-releasing activity of injected InsP3. The average amplitude of the Ca2+ peak seen in control eggs (0.55±0.04 arbitrary units) was strongly reduced (0.25±0.05), but not completely abolished (Fig. 3). The significance of this observation will be discussed in more detail later on (see Discussion).

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