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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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During fertilization, the intracellular Ca2+ release is followed by the biphasic increase of PIP2 at the plasma membrane.Mature eggs loaded with Calcium Green were microinjected with RFP-PH (150 µM, pipette concentration) to monitor the changes of PIP2 and sperm-induced Ca2+ signals. A representative result from 15 independent experiments was presented. (A) Changes of RFP-PH signals at the site of fertilization. The moment the spermatozoon is stopped at the egg surface was set to t = 0:00 (min:sec). The onset of PIP2 increment is evident by 0:36 (arrows). (B) Images of instantaneous increment (Finst = [(Ft-Ft−1)/Ft−1]) in Ca2+ release (green) and PIP2 (red) were merged with the light transmission photomicrographs of the fertilized egg at the corresponding time points. The position of the fertilizing sperm was indicated with an arrow. (C) Temporal relationship between sperm-induced Ca2+ signaling (Frel = [F−F0]/F0; green curve) and the fluctuation of the relative fluorescence (Frel = [Fpm−Fct]/Fct; red curve) for plasma membrane PIP2 in the region of the framed area of the sperm-egg interaction.
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pone-0014100-g002: During fertilization, the intracellular Ca2+ release is followed by the biphasic increase of PIP2 at the plasma membrane.Mature eggs loaded with Calcium Green were microinjected with RFP-PH (150 µM, pipette concentration) to monitor the changes of PIP2 and sperm-induced Ca2+ signals. A representative result from 15 independent experiments was presented. (A) Changes of RFP-PH signals at the site of fertilization. The moment the spermatozoon is stopped at the egg surface was set to t = 0:00 (min:sec). The onset of PIP2 increment is evident by 0:36 (arrows). (B) Images of instantaneous increment (Finst = [(Ft-Ft−1)/Ft−1]) in Ca2+ release (green) and PIP2 (red) were merged with the light transmission photomicrographs of the fertilized egg at the corresponding time points. The position of the fertilizing sperm was indicated with an arrow. (C) Temporal relationship between sperm-induced Ca2+ signaling (Frel = [F−F0]/F0; green curve) and the fluctuation of the relative fluorescence (Frel = [Fpm−Fct]/Fct; red curve) for plasma membrane PIP2 in the region of the framed area of the sperm-egg interaction.

Mentions: Mature eggs of A. aranciacus were loaded with RFP-PH and Calcium Green and inseminated to monitor the changes in PIP2 and cytosolic Ca2+ levels at the time of fertilization. By 36 sec after the sperm-egg contact, the labeling of PIP2 at the egg plasma membrane was markedly increased near the site of sperm interaction (Fig. 2A, white arrows). At this time, the sperm head was still located outside the jelly coat (Fig. 2B, arrow). When the data were analyzed for the incremental changes of the Ca2+ and PIP2 levels to focalize the sites of instantaneous rises by applying the formula (Finst = [(Ft-Ft−1)/Ft−1]), it was evident that the initial increase of PIP2 took place at the sperm interaction site 9 sec after the sperm-induced Ca2+ release (see the images at 0:27 and 0:36 in Fig. 2B). When evaluated in reference to the level in the cytoplasm, the plasma membrane PIP2 levels displayed a dual response to the fertilizing sperm: the quick initial rise and fall and the prolonged late increase (Fig. 2C). The relative level of PIP2 at the plasma membrane (Frel  = [Fp-Fc]/Fc) increased after fertilization, but it took place nearly 10 sec after the onset to the Ca2+ signaling (Fig. 2C, arrow). After the sharp increase of Ca2+ over a certain level (>0.3 R.F. unit), the relative level of PIP2 at the plasma membrane (Frel values) began to decrease at 40 sec (Fig. 2C, arrow) and continued to subside to the negative numbers until about 60 sec, implying that much of the membrane-bound RFP-PH was translocated to the cytoplasm. This period of steep decrease of the RFP-PH signals at the plasma membrane corresponds to 80–100 sec after the addition of sperm, and the result is in general agreement with the timing of the accelerated InsP3 production observed in the fertilized eggs of the sea urchin species such as A. crassispina, H. pulcherrimus, and L. pictus [29], [32]. Subsequently, however, the Frel values of plasma membrane PIP2 of the starfish egg gradually increased again (Fig. 2C, asterisk) and remained elevated for five min after fertilization.


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)

During fertilization, the intracellular Ca2+ release is followed by the biphasic increase of PIP2 at the plasma membrane.Mature eggs loaded with Calcium Green were microinjected with RFP-PH (150 µM, pipette concentration) to monitor the changes of PIP2 and sperm-induced Ca2+ signals. A representative result from 15 independent experiments was presented. (A) Changes of RFP-PH signals at the site of fertilization. The moment the spermatozoon is stopped at the egg surface was set to t = 0:00 (min:sec). The onset of PIP2 increment is evident by 0:36 (arrows). (B) Images of instantaneous increment (Finst = [(Ft-Ft−1)/Ft−1]) in Ca2+ release (green) and PIP2 (red) were merged with the light transmission photomicrographs of the fertilized egg at the corresponding time points. The position of the fertilizing sperm was indicated with an arrow. (C) Temporal relationship between sperm-induced Ca2+ signaling (Frel = [F−F0]/F0; green curve) and the fluctuation of the relative fluorescence (Frel = [Fpm−Fct]/Fct; red curve) for plasma membrane PIP2 in the region of the framed area of the sperm-egg interaction.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014100-g002: During fertilization, the intracellular Ca2+ release is followed by the biphasic increase of PIP2 at the plasma membrane.Mature eggs loaded with Calcium Green were microinjected with RFP-PH (150 µM, pipette concentration) to monitor the changes of PIP2 and sperm-induced Ca2+ signals. A representative result from 15 independent experiments was presented. (A) Changes of RFP-PH signals at the site of fertilization. The moment the spermatozoon is stopped at the egg surface was set to t = 0:00 (min:sec). The onset of PIP2 increment is evident by 0:36 (arrows). (B) Images of instantaneous increment (Finst = [(Ft-Ft−1)/Ft−1]) in Ca2+ release (green) and PIP2 (red) were merged with the light transmission photomicrographs of the fertilized egg at the corresponding time points. The position of the fertilizing sperm was indicated with an arrow. (C) Temporal relationship between sperm-induced Ca2+ signaling (Frel = [F−F0]/F0; green curve) and the fluctuation of the relative fluorescence (Frel = [Fpm−Fct]/Fct; red curve) for plasma membrane PIP2 in the region of the framed area of the sperm-egg interaction.
Mentions: Mature eggs of A. aranciacus were loaded with RFP-PH and Calcium Green and inseminated to monitor the changes in PIP2 and cytosolic Ca2+ levels at the time of fertilization. By 36 sec after the sperm-egg contact, the labeling of PIP2 at the egg plasma membrane was markedly increased near the site of sperm interaction (Fig. 2A, white arrows). At this time, the sperm head was still located outside the jelly coat (Fig. 2B, arrow). When the data were analyzed for the incremental changes of the Ca2+ and PIP2 levels to focalize the sites of instantaneous rises by applying the formula (Finst = [(Ft-Ft−1)/Ft−1]), it was evident that the initial increase of PIP2 took place at the sperm interaction site 9 sec after the sperm-induced Ca2+ release (see the images at 0:27 and 0:36 in Fig. 2B). When evaluated in reference to the level in the cytoplasm, the plasma membrane PIP2 levels displayed a dual response to the fertilizing sperm: the quick initial rise and fall and the prolonged late increase (Fig. 2C). The relative level of PIP2 at the plasma membrane (Frel  = [Fp-Fc]/Fc) increased after fertilization, but it took place nearly 10 sec after the onset to the Ca2+ signaling (Fig. 2C, arrow). After the sharp increase of Ca2+ over a certain level (>0.3 R.F. unit), the relative level of PIP2 at the plasma membrane (Frel values) began to decrease at 40 sec (Fig. 2C, arrow) and continued to subside to the negative numbers until about 60 sec, implying that much of the membrane-bound RFP-PH was translocated to the cytoplasm. This period of steep decrease of the RFP-PH signals at the plasma membrane corresponds to 80–100 sec after the addition of sperm, and the result is in general agreement with the timing of the accelerated InsP3 production observed in the fertilized eggs of the sea urchin species such as A. crassispina, H. pulcherrimus, and L. pictus [29], [32]. Subsequently, however, the Frel values of plasma membrane PIP2 of the starfish egg gradually increased again (Fig. 2C, asterisk) and remained elevated for five min after fertilization.

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
Related in: MedlinePlus