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The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.

Chun JT, Puppo A, Vasilev F, Gragnaniello G, Garante E, Santella L - PLoS ONE (2010)

Bottom Line: The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact.Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization.

View Article: PubMed Central - PubMed

Affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy. chun@szn.it

ABSTRACT

Background: Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+). Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3)) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.

Methodology/principal findings: In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca(2+) and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+) wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.

Conclusions/significance: Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.

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Perivitelline spike formation is not limited to starfish eggs, but is also present in sea urchin eggs at fertilization.Starfish (A. pectinifera) (A) and sea urchin (P. lividus) eggs (B) were stained with FM 1-43 as described in Materials and Methods. After immediate rinse with FSW, sperm were added (t = 0), and the eggs were imaged with confocal microscopy to monitor the spike formation and the elevation of the fertilization envelope.
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pone-0014100-g009: Perivitelline spike formation is not limited to starfish eggs, but is also present in sea urchin eggs at fertilization.Starfish (A. pectinifera) (A) and sea urchin (P. lividus) eggs (B) were stained with FM 1-43 as described in Materials and Methods. After immediate rinse with FSW, sperm were added (t = 0), and the eggs were imaged with confocal microscopy to monitor the spike formation and the elevation of the fertilization envelope.

Mentions: To test if the spike formation in the perivitelline space is limited to a certain species of starfish or to a detection method (RFP-PH) (Fig. 3A), we visualized the perivitelline spikes in an alternative method. To this end, eggs of a different starfish species (Asterina pectinifera) and sea urchin (Paracentrotus lividus) were briefly stained prior to fertilization, by use of a lipophilic dye that selectively delineates the plasma membrane. As shown in Fig. 9, the egg activation after fertilization was much faster in sea urchin eggs. By 3 min after insemination, the fertilization envelope was fully elevated, and a myriad of spikes were visualized by FM 1-43 in the perivitelline space (Fig. 9B). In the same time scale, the starfish eggs were activated more slowly but displayed a similar pattern of spike formation in the perivitelline space (Fig. 9A).


The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.

Chun JT, Puppo A, Vasilev F, Gragnaniello G, Garante E, Santella L - PLoS ONE (2010)

Perivitelline spike formation is not limited to starfish eggs, but is also present in sea urchin eggs at fertilization.Starfish (A. pectinifera) (A) and sea urchin (P. lividus) eggs (B) were stained with FM 1-43 as described in Materials and Methods. After immediate rinse with FSW, sperm were added (t = 0), and the eggs were imaged with confocal microscopy to monitor the spike formation and the elevation of the fertilization envelope.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990714&req=5

pone-0014100-g009: Perivitelline spike formation is not limited to starfish eggs, but is also present in sea urchin eggs at fertilization.Starfish (A. pectinifera) (A) and sea urchin (P. lividus) eggs (B) were stained with FM 1-43 as described in Materials and Methods. After immediate rinse with FSW, sperm were added (t = 0), and the eggs were imaged with confocal microscopy to monitor the spike formation and the elevation of the fertilization envelope.
Mentions: To test if the spike formation in the perivitelline space is limited to a certain species of starfish or to a detection method (RFP-PH) (Fig. 3A), we visualized the perivitelline spikes in an alternative method. To this end, eggs of a different starfish species (Asterina pectinifera) and sea urchin (Paracentrotus lividus) were briefly stained prior to fertilization, by use of a lipophilic dye that selectively delineates the plasma membrane. As shown in Fig. 9, the egg activation after fertilization was much faster in sea urchin eggs. By 3 min after insemination, the fertilization envelope was fully elevated, and a myriad of spikes were visualized by FM 1-43 in the perivitelline space (Fig. 9B). In the same time scale, the starfish eggs were activated more slowly but displayed a similar pattern of spike formation in the perivitelline space (Fig. 9A).

Bottom Line: The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact.Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization.

View Article: PubMed Central - PubMed

Affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy. chun@szn.it

ABSTRACT

Background: Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+). Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3)) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.

Methodology/principal findings: In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca(2+) and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+) wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.

Conclusions/significance: Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.

Show MeSH