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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

The spatiotemporal relationship among sperm entry, Ca2+ release, and the elevation of the vitelline layer in fertilized starfish eggs.(A) The transmission images of a representative fertilized egg (A. aranciacus) were superimposed with the corresponding fluorograms of the Ca2+ indicator at each time point. The moment of sperm's arrival at the jelly coat was set to t = 0:00 (min:sec). Following the quick cortical flash at 0:26 (arrow), a massive Ca2+ wave initiated from the sperm entry site (0:30) and propagated to the opposite side of the egg. At 3:03 when the Ca2+ wave had already encroached upon the entire cytoplasm, the fertilization envelope began to be elevated (arrow). The fertilization envelope was fully formed only after the Ca2+ wave had traversed the entire cytoplasm at 6:40 (arrow). (B) Detailed views of the sperm entry site (the area marked by a small blue rectangle in panel A) during fertilization. At 2:04 when the vitelline layer is locally elevated, the sperm is still located in the jelly coat (arrow). At 4:00, the sperm head still remains on the outside surface of the egg, but the long acrosomal process (arrow) is inside the egg and connected to a filamentous structure (arrowheads). At 4:42, the sperm head is still visible (arrow), the vitelline layer is further elevated and the focal plasma membrane at the sperm entry site is now being detached from the vitelline layer. At 4:42 and at 6:40, the sperm head (arrow) is still inside the jelly coat. At 7:01, the sperm head is inside the egg cytoplasm (arrow), and the plasma membrane is retracting behind the sperm. The tail is still outside (arrow). At 8:22 and at 9:44, the tail of the sperm finally enters the egg cytoplasm (arrowhead), as the plasma membrane further seals to form a fertilization cone. The motion picture of the entire process is available as a video file (Data S1).
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pone-0003588-g001: The spatiotemporal relationship among sperm entry, Ca2+ release, and the elevation of the vitelline layer in fertilized starfish eggs.(A) The transmission images of a representative fertilized egg (A. aranciacus) were superimposed with the corresponding fluorograms of the Ca2+ indicator at each time point. The moment of sperm's arrival at the jelly coat was set to t = 0:00 (min:sec). Following the quick cortical flash at 0:26 (arrow), a massive Ca2+ wave initiated from the sperm entry site (0:30) and propagated to the opposite side of the egg. At 3:03 when the Ca2+ wave had already encroached upon the entire cytoplasm, the fertilization envelope began to be elevated (arrow). The fertilization envelope was fully formed only after the Ca2+ wave had traversed the entire cytoplasm at 6:40 (arrow). (B) Detailed views of the sperm entry site (the area marked by a small blue rectangle in panel A) during fertilization. At 2:04 when the vitelline layer is locally elevated, the sperm is still located in the jelly coat (arrow). At 4:00, the sperm head still remains on the outside surface of the egg, but the long acrosomal process (arrow) is inside the egg and connected to a filamentous structure (arrowheads). At 4:42, the sperm head is still visible (arrow), the vitelline layer is further elevated and the focal plasma membrane at the sperm entry site is now being detached from the vitelline layer. At 4:42 and at 6:40, the sperm head (arrow) is still inside the jelly coat. At 7:01, the sperm head is inside the egg cytoplasm (arrow), and the plasma membrane is retracting behind the sperm. The tail is still outside (arrow). At 8:22 and at 9:44, the tail of the sperm finally enters the egg cytoplasm (arrowhead), as the plasma membrane further seals to form a fertilization cone. The motion picture of the entire process is available as a video file (Data S1).

Mentions: The fertilizing spermatozoon induces a massive release of Ca2+ inside the egg, which leads to the exocytosis of cortical granules and to the subsequent elevation of the vitelline layer [7], [12], [37], [38]. The precise spatiotemporal relationship that exists for sperm entry, Ca2+ entry, and the formation of the fertilization envelope has not been described in detail. Owing to the large cell size and optical transparency of the cytoplasm, starfish eggs have been an excellent model system for studying intracellular Ca2+ release and morphological changes with the help of fluorescent Ca2+ indicators and imaging systems [6]. As shown in Fig. 1A, sperm addition to the A. aranciacus egg produced the characteristic cortical Ca2+ flash and the propagation of the Ca2+ wave from the site of sperm-egg interaction. We have noticed that the fertilizing sperm is able to induce Ca2+ signaling and to initiate the vitelline layer elevation while still outside the egg (arrow in Fig. 1A, at 3:03). One minute after the initiation of the Ca2+ burst, the vitelline layer was further elevated, but the sperm was still outside the egg. However, the sperm had inserted its long acrosomal process in the egg cytoplasm (arrow in Fig. 1B, at 4:00), forming a fertilization cone. Curiously, the acrosomal process appeared prolonged inside the egg by the conjunction with filamentous structure inside the egg (arrowheads in Fig. 1B, at 4:00). The vitelline layer became further elevated with time, while the sperm was still located in the jelly coat outside the egg. At this point, the plasma membrane of the egg was not yet detached from the vitelline layer in the fertilization cone. Only after this portion of the egg plasma membrane had become detached from the elevating vitelline layer did the sperm physically enter the egg cytoplasm (Fig. 1B, at 7:01). At 4:42 the sperm head was still visible as it approached the egg plasma membrane (arrow). After it had crossed it (at 7:01) it became lost among the other cytoplasmic densities (arrow), only its tail still being visible outside (arrowhead). As the vitelline layer was fully elevated, the sperm tail also entered the egg cytoplasm (Fig. 1B, at 8:22 and at 9:44, arrowheads). At the conclusion of the process, the egg plasma membrane at the sperm entry site sealed with the formation of a round bleb (Fig. 1B, at 13:53). The entire process is presented as a video file in Data S1.


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)

The spatiotemporal relationship among sperm entry, Ca2+ release, and the elevation of the vitelline layer in fertilized starfish eggs.(A) The transmission images of a representative fertilized egg (A. aranciacus) were superimposed with the corresponding fluorograms of the Ca2+ indicator at each time point. The moment of sperm's arrival at the jelly coat was set to t = 0:00 (min:sec). Following the quick cortical flash at 0:26 (arrow), a massive Ca2+ wave initiated from the sperm entry site (0:30) and propagated to the opposite side of the egg. At 3:03 when the Ca2+ wave had already encroached upon the entire cytoplasm, the fertilization envelope began to be elevated (arrow). The fertilization envelope was fully formed only after the Ca2+ wave had traversed the entire cytoplasm at 6:40 (arrow). (B) Detailed views of the sperm entry site (the area marked by a small blue rectangle in panel A) during fertilization. At 2:04 when the vitelline layer is locally elevated, the sperm is still located in the jelly coat (arrow). At 4:00, the sperm head still remains on the outside surface of the egg, but the long acrosomal process (arrow) is inside the egg and connected to a filamentous structure (arrowheads). At 4:42, the sperm head is still visible (arrow), the vitelline layer is further elevated and the focal plasma membrane at the sperm entry site is now being detached from the vitelline layer. At 4:42 and at 6:40, the sperm head (arrow) is still inside the jelly coat. At 7:01, the sperm head is inside the egg cytoplasm (arrow), and the plasma membrane is retracting behind the sperm. The tail is still outside (arrow). At 8:22 and at 9:44, the tail of the sperm finally enters the egg cytoplasm (arrowhead), as the plasma membrane further seals to form a fertilization cone. The motion picture of the entire process is available as a video file (Data S1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003588-g001: The spatiotemporal relationship among sperm entry, Ca2+ release, and the elevation of the vitelline layer in fertilized starfish eggs.(A) The transmission images of a representative fertilized egg (A. aranciacus) were superimposed with the corresponding fluorograms of the Ca2+ indicator at each time point. The moment of sperm's arrival at the jelly coat was set to t = 0:00 (min:sec). Following the quick cortical flash at 0:26 (arrow), a massive Ca2+ wave initiated from the sperm entry site (0:30) and propagated to the opposite side of the egg. At 3:03 when the Ca2+ wave had already encroached upon the entire cytoplasm, the fertilization envelope began to be elevated (arrow). The fertilization envelope was fully formed only after the Ca2+ wave had traversed the entire cytoplasm at 6:40 (arrow). (B) Detailed views of the sperm entry site (the area marked by a small blue rectangle in panel A) during fertilization. At 2:04 when the vitelline layer is locally elevated, the sperm is still located in the jelly coat (arrow). At 4:00, the sperm head still remains on the outside surface of the egg, but the long acrosomal process (arrow) is inside the egg and connected to a filamentous structure (arrowheads). At 4:42, the sperm head is still visible (arrow), the vitelline layer is further elevated and the focal plasma membrane at the sperm entry site is now being detached from the vitelline layer. At 4:42 and at 6:40, the sperm head (arrow) is still inside the jelly coat. At 7:01, the sperm head is inside the egg cytoplasm (arrow), and the plasma membrane is retracting behind the sperm. The tail is still outside (arrow). At 8:22 and at 9:44, the tail of the sperm finally enters the egg cytoplasm (arrowhead), as the plasma membrane further seals to form a fertilization cone. The motion picture of the entire process is available as a video file (Data S1).
Mentions: The fertilizing spermatozoon induces a massive release of Ca2+ inside the egg, which leads to the exocytosis of cortical granules and to the subsequent elevation of the vitelline layer [7], [12], [37], [38]. The precise spatiotemporal relationship that exists for sperm entry, Ca2+ entry, and the formation of the fertilization envelope has not been described in detail. Owing to the large cell size and optical transparency of the cytoplasm, starfish eggs have been an excellent model system for studying intracellular Ca2+ release and morphological changes with the help of fluorescent Ca2+ indicators and imaging systems [6]. As shown in Fig. 1A, sperm addition to the A. aranciacus egg produced the characteristic cortical Ca2+ flash and the propagation of the Ca2+ wave from the site of sperm-egg interaction. We have noticed that the fertilizing sperm is able to induce Ca2+ signaling and to initiate the vitelline layer elevation while still outside the egg (arrow in Fig. 1A, at 3:03). One minute after the initiation of the Ca2+ burst, the vitelline layer was further elevated, but the sperm was still outside the egg. However, the sperm had inserted its long acrosomal process in the egg cytoplasm (arrow in Fig. 1B, at 4:00), forming a fertilization cone. Curiously, the acrosomal process appeared prolonged inside the egg by the conjunction with filamentous structure inside the egg (arrowheads in Fig. 1B, at 4:00). The vitelline layer became further elevated with time, while the sperm was still located in the jelly coat outside the egg. At this point, the plasma membrane of the egg was not yet detached from the vitelline layer in the fertilization cone. Only after this portion of the egg plasma membrane had become detached from the elevating vitelline layer did the sperm physically enter the egg cytoplasm (Fig. 1B, at 7:01). At 4:42 the sperm head was still visible as it approached the egg plasma membrane (arrow). After it had crossed it (at 7:01) it became lost among the other cytoplasmic densities (arrow), only its tail still being visible outside (arrowhead). As the vitelline layer was fully elevated, the sperm tail also entered the egg cytoplasm (Fig. 1B, at 8:22 and at 9:44, arrowheads). At the conclusion of the process, the egg plasma membrane at the sperm entry site sealed with the formation of a round bleb (Fig. 1B, at 13:53). The entire process is presented as a video file in Data S1.

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