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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.

Show MeSH
Heparin blocks sperm entry.The fertilization process in the heparin-injected egg was monitored with a CCD camera, and the key moments were presented by still-shot photomicrographs. The moment of sperm attachment to the egg surface was set to t = 0:00 (min:sec). At 0:41, the sperm was still attached to the jelly coat (arrow). Afterwards, the sperm attempts but fails to penetrate the jelly coat. At 5:20, the vitelline layer is visibly elevated, but the sperm is still completely outside the jelly coat. The formation of the fertilization cone is evident under the elevating membrane (arrow). At 7:52, the vitelline layer is further elevated, but the fertilization cone fails to pull in the sperm head. The fertilization envelope is now being established while the sperm is still outside. The motion picture of the entire process is available as a video file in Data S3.
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pone-0003588-g008: Heparin blocks sperm entry.The fertilization process in the heparin-injected egg was monitored with a CCD camera, and the key moments were presented by still-shot photomicrographs. The moment of sperm attachment to the egg surface was set to t = 0:00 (min:sec). At 0:41, the sperm was still attached to the jelly coat (arrow). Afterwards, the sperm attempts but fails to penetrate the jelly coat. At 5:20, the vitelline layer is visibly elevated, but the sperm is still completely outside the jelly coat. The formation of the fertilization cone is evident under the elevating membrane (arrow). At 7:52, the vitelline layer is further elevated, but the fertilization cone fails to pull in the sperm head. The fertilization envelope is now being established while the sperm is still outside. The motion picture of the entire process is available as a video file in Data S3.

Mentions: As mentioned earlier, alteration of the cortical actin cytoskeleton by heparin leads to the polyspermic formation of fertilization cones. To test if heparin also affected sperm entry, the fertilization process was monitored with a CCD camera. Although heparin-treated eggs produced at least 5 times more fertilization cones than control eggs in a given focal plane, the frequency of actual sperm entry through these fertilization cones was significantly lower. Whereas 9 out of 10 fertilization cones in the focal plane of control eggs exhibited successful sperm entry, the success rate in the heparin-treated eggs was 14 out of 28 (n = 8). Similar results were obtained when sperm were prestained with the DNA dye Hoechst 33342 (not shown). The increased occurrence of ‘empty’ fertilization cones in heparin-treated eggs enabled us to monitor the abortive procedure of sperm entry. In the heparin-treated eggs, sperm initially penetrated the jelly coat (Fig. 8, see the position of the sperm head at 0:41 and at 2:51) in a way similar to the control, but the continuous movement of the elevating vitelline layer failed to ‘pull’ the sperm into the egg cytoplasm. The vitelline layer continued to elevate with the formation of the ‘empty’ fertilization cones (Fig. 8, arrow at 5:20), but in most cases the sperm was apparently pushed back to the jelly coat (see the video file in Data S3).


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 blocks sperm entry.The fertilization process in the heparin-injected egg was monitored with a CCD camera, and the key moments were presented by still-shot photomicrographs. The moment of sperm attachment to the egg surface was set to t = 0:00 (min:sec). At 0:41, the sperm was still attached to the jelly coat (arrow). Afterwards, the sperm attempts but fails to penetrate the jelly coat. At 5:20, the vitelline layer is visibly elevated, but the sperm is still completely outside the jelly coat. The formation of the fertilization cone is evident under the elevating membrane (arrow). At 7:52, the vitelline layer is further elevated, but the fertilization cone fails to pull in the sperm head. The fertilization envelope is now being established while the sperm is still outside. The motion picture of the entire process is available as a video file in Data S3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003588-g008: Heparin blocks sperm entry.The fertilization process in the heparin-injected egg was monitored with a CCD camera, and the key moments were presented by still-shot photomicrographs. The moment of sperm attachment to the egg surface was set to t = 0:00 (min:sec). At 0:41, the sperm was still attached to the jelly coat (arrow). Afterwards, the sperm attempts but fails to penetrate the jelly coat. At 5:20, the vitelline layer is visibly elevated, but the sperm is still completely outside the jelly coat. The formation of the fertilization cone is evident under the elevating membrane (arrow). At 7:52, the vitelline layer is further elevated, but the fertilization cone fails to pull in the sperm head. The fertilization envelope is now being established while the sperm is still outside. The motion picture of the entire process is available as a video file in Data S3.
Mentions: As mentioned earlier, alteration of the cortical actin cytoskeleton by heparin leads to the polyspermic formation of fertilization cones. To test if heparin also affected sperm entry, the fertilization process was monitored with a CCD camera. Although heparin-treated eggs produced at least 5 times more fertilization cones than control eggs in a given focal plane, the frequency of actual sperm entry through these fertilization cones was significantly lower. Whereas 9 out of 10 fertilization cones in the focal plane of control eggs exhibited successful sperm entry, the success rate in the heparin-treated eggs was 14 out of 28 (n = 8). Similar results were obtained when sperm were prestained with the DNA dye Hoechst 33342 (not shown). The increased occurrence of ‘empty’ fertilization cones in heparin-treated eggs enabled us to monitor the abortive procedure of sperm entry. In the heparin-treated eggs, sperm initially penetrated the jelly coat (Fig. 8, see the position of the sperm head at 0:41 and at 2:51) in a way similar to the control, but the continuous movement of the elevating vitelline layer failed to ‘pull’ the sperm into the egg cytoplasm. The vitelline layer continued to elevate with the formation of the ‘empty’ fertilization cones (Fig. 8, arrow at 5:20), but in most cases the sperm was apparently pushed back to the jelly coat (see the video file in Data S3).

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