Limatola N et al. (2022), Regulation of the Actin Cytoskeleton-Linked Ca2...

ECB-ART-50333

Cells 2022 Apr 29;119:. doi: 10.3390/cells11091496.

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Regulation of the Actin Cytoskeleton-Linked Ca2+ Signaling by Intracellular pH in Fertilized Eggs of Sea Urchin.

Limatola N , Chun JT , Santella L .

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In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg's receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process.

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	Figure 1. Effect of ammonia seawater on the outer surface of unfertilized P. lividus eggs. Before fixation, P. lividus eggs of the same batch were partitioned and incubated for 20 min either in NSW at pH 8.1 (control) or in SW adjusted to pH 9 using NH4OH (ammonia seawater). Changes in the outer egg surface and cortical ultrastructure were observed by SEM (A–D) or TEM (E,F). Note the eggs’ undulated surface (B) and microvilli’s changes in shape and length (D). Microvilli covered by the vitelline layer are relatively uniform on the surface of the control egg (C,E) but are often elongated on the surface of eggs incubated in ammonia seawater (D,F). Abbreviations: VL, vitelline layer; MV, microvilli; CG, cortical granules.
	Figure 2. Sea urchin eggs fertilized in ammonia seawater display altered Ca2+ response. P. lividus eggs were microinjected with the calcium dye and incubated for 20 min either in NSW pH 8.1. (control) or in ammonia seawater. Then, the sperm-induced Ca2+ increases in these eggs were compared. (A) Momentary increases in Ca2+ levels were visualized in pseudocolor in the control egg (fertilized in NSW) and in the egg fertilized in ammonia seawater (B). The time point showing cortical flash (CF, indicated by an arrow) was set as t = 0. The arrowheads indicate the sperm interaction sites on the egg surface and the Ca2+ waves triggered by two sperm. (C) The trajectory of the intracellular Ca2+ levels in control eggs (green curves) and the eggs fertilized in ammonia seawater (brown curves). The vertical arrow indicates the CF, and the arrowhead indicates the more abruptly decaying intracellular Ca2+ levels 300 s after insemination. The inset shows the same Ca2+ trajectories plotted on a different time scale to visualize the initial Ca2+ changes more clearly. (D) Histograms showing the most affected aspects of Ca2+ signal at fertilization: amplitude of the cortical flash (CF) and declining kinetics of the Ca2+ wave expressed in terms of the time required for the Ca2+ level RFU to reach 0.2 RFU. Tukey HSD test, p < 0.01. Abbreviation: RFU, relative fluorescence unit.
	Figure 3. Effect of ammonia seawater on the egg receptivity to sperm. P. lividus eggs pre-incubated for 20 min in NSW (pH 8.1) (A) or in ammonia seawater (pH 9) were fertilized by Hoechst 33342-stained sperm (B). The number of sperm in the living zygote was counted 5 min after insemination by a CCD camera with a UV laser in epifluorescence microscopy (middle panel). The elevation of the fertilization envelope (FE) was visualized in the bright field view, and the merged image was provided to distinguish sperm inside the egg from those attached to the FE.
	Figure 4. Effect of ammonia seawater on F-actin dynamics in fertilized eggs. P. lividus eggs were microinjected with AlexaFluor568-phalloidin and incubated for 20 min in normal seawater (NSW, pH 8.1) (A) or in ammonia seawater (pH 9) prior to fertilization in the same medium (B). The structural changes in the actin cytoskeleton following fertilization were monitored with the laser scanning confocal microscope. The moment of sperm addition was set as t = 0.
	Figure 5. Surface topography and cortical ultrastructure of the eggs fertilized in ammonia seawater. P. lividus eggs were fertilized and fixed 5 min after insemination for SEM (A–D) and TEM (E,F) observations. (A,C) Control eggs fertilized in NSW at pH 8.1. (B,D) Eggs were pre-incubated (20 min) and fertilized in ammonia seawater at pH 9. Enlarged view of the elevated FE covering the control egg (C) and the punctured FE of the egg fertilized in ammonia seawater (D). Note in panel D the over-elongated microvilli passing through the punctured FE. Abbreviations: FE, fertilization envelope; HL, hyaline layer; MV, microvilli.
	Figure 6. Abnormal development of the eggs fertilized following alkalinization of their cytoplasm. P. lividus eggs were fertilized after incubation in normal seawater (NSW, pH 8.1) (A) or ammonia seawater (pH 9) (B), and then the subsequent development was monitored by light microscopy after 3 hr. Note that the FE, which had undergone elevation in the eggs incubated and fertilized in ammonia seawater, is seen collapsed on the surface of embryos after 3 h of insemination.

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Begg, pH regulates the polymerization of actin in the sea urchin egg cortex. 1979, Pubmed, Echinobase