Vasilev F et al. (2019), Contributions of suboolemmal acidic vesicles an...

ECB-ART-47069

Int J Biol Sci 2019 Jan 29;154:757-775. doi: 10.7150/ijbs.28461.

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Contributions of suboolemmal acidic vesicles and microvilli to the intracellular Ca2+ increase in the sea urchin eggs at fertilization.

Vasilev F , Limatola N , Chun JT , Santella L .

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The onset of fertilization in echinoderms is characterized by instantaneous increase of Ca2+ in the egg cortex, which is called ''cortical flash'', and the subsequent Ca2+ wave. While the cortical flash is due to the ion influx through L-type Ca2+ channels in starfish eggs, its amplitude was shown to be affected by the integrity of the egg cortex. Here, we investigated the contribution of cortical granules (CG) and yolk granules (YG) to the sperm-induced Ca2+ signals in sea urchin eggs. To this end, prior to fertilization, Paracentrotus lividus eggs were treated with agents that disrupt or relocate CG beneath the plasma membrane: namely, glycyl-L-phenylalanine 2-naphthylamide (GPN), procaine, urethane, and NH4Cl. All these pretreatments consistently suppressed the cortical flash in the fertilized eggs, and accelerated the decay kinetics of the subsiding Ca2+ wave in most cases. By contrast, centrifugation of the eggs, which stratifies organelles but not the CG, did not exhibit such changes except that the CF was much enhanced in the centrifugal pole where YG are localized. Surprisingly, we noted that pretreatment of the eggs with these CG-disrupting agents or with the inhibitors of L-type Ca2+ channels all drastically reduced the density of the microvilli and their individual shapes on the egg surface. Taken together, our results suggest that the integrity of the egg cortex ensures successful generation of the Ca2+ responses at fertilization, and that modulation of microvilli shape and density may serve as a mechanism of controlling ion flux across the plasma membrane.

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Genes referenced: LOC100887844 LOC115919910 pole stk36

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	Figure 1. The cortical flash depends on the Ca2+ influx via L-type Ca2+ channels. (A) The eggs fertilized in the ASW with 1 mM Ca2+ (brown curves) displayed virtually the same Ca2+ response as in control eggs (ASW with 10 mM Ca2+, green curves), but the CFs were severely suppressed. (B-C) P. lividus eggs were pretreated with either diltiazem or verapamil for 40 minutes, and their Ca2+ responses at fertilization were compared with that of control eggs (FSW) in the same batch. The trajectories of the Ca2+ levels in the fertilized eggs that had been pretreated with 50 µM diltiazem or 10 µM verapamil were shown in brown curves, whereas the responses in the control eggs were presented in green curves. (D) The dose-dependent effects of diltiazem and verapamil on the Ca2+ signalling in fertilized eggs. The peak amplitudes of the CF (red line) and the global Ca2+ increase (blue line) in the eggs pretreated with varied amount of diltiazem and verapamil were normalized with the corresponding average levels in the control eggs of the same batch.
	Figure 2. Fertilization of the sea urchin eggs pretreated with GPN. P. lividus eggs were fertilized following the pretreatments described in the Materials and Methods. (A) Pseudocolor images of the instantaneous changes of the Ca2+ signals at several key moments of the propagating wave in representative eggs. The arrow indicates the intracellular Ca2+ increase at the spot of the sperm-egg interaction site. (B) The quantified Ca2+ increases in the control (green curves) and GPN-pretreated (200 µM, brown curves) eggs in one of the four independent experiments. (C) Dose-dependent effects of GPN on the elevation of the FE and polyspermy. Sperm inside the fertilized eggs were visualized with the Hoechst 33342 (arrows), scale bar 20 µm. (D) The average numbers of the egg-incorporated spermatozoa were provided as Mean ± SD (bars) and the FE elevation as a frequency (line).
	Figure 3. Effect of GPN pretreatment on the CG and vesicles. (A) P. lividus eggs were stained with LysoTracker-RED before and after (40 min) the treatment with 200 µM GPN or DMSO (control), and viewed in confocal microscopy. The changes of the fluorescent signals in the LysoTracker-RED-stained vesicles were examined in the same eggs 25 minutes after fertilization. Control eggs displayed full elevation of the FE, but most eggs treated with GPN failed at the FE elevation despite the formation of multiple fertilization cones (blue arrowheads). (B) Electron micrographs of the cortex of the eggs treated with 200 µM GPN or DMSO. In control eggs, cortical granules (red arrow) positioned underneath the plasma membrane were all exocytosed into the perivitelline space 5 minutes after fertilization (right panel). In GPN-pretreated eggs, CG appeared to fuse with each other (white arrow) or with the adjacent vesicles (black arrow), and to be displaced from the plasma membrane. After insemination, FE elevates only partially while some CG fused with other granules (black arrow) are still visible inside the eggs.
	Figure 4. Ultrastructural changes of the egg surface after the treatment with GPN, diltiazem and verapamil. (A) SEM images showing the microvilli of the unfertilized eggs treated with GPN (200 µM, 40 min) and DMSO. Note the reduced density of microvilli and their elongated shape in GPN-treated eggs in comparison with the control egg exhibiting regularly dispersed and shorter microvilli. (B) Effects of verapamil (50 µM, 40 min) and diltiazem (50 µM, 40 min) on the microvilli structure and quantity. Whereas SEM images of the control egg (FSW) display microvilli of regular distribution and length, the microvilli of the eggs treated with the two L-type Ca2+ channel inhibitors show irregular shapes and reduced quantity (also see Table 1). Scale bar 1 µm.
	Figure 5. Effects of urethane, procaine and NH4Cl on the ultrastucture of the egg surface. (A) TEM and SEM images of P. lividus control eggs show dense microvilli with nearly uniform shape and size. In the TEM image (left panel), CG are apparently attached to the plasma membrane. (B) After urethane treatment (400 mM, 5 min), egg surface shows a sign of corrugation and significant loss of microvilli (SEM). The treatment induces invaginations on the egg surface and dislocation of some CG from the plasma membrane (TEM, black arrow). (C) Incubation with procaine (10 mM, 20 min) gives rise to microvilli elongation as well as reduction in their number. Some parts of the cell surface are deprived of microvilli (SEM, right panel). Procaine-pretreated eggs show bulges on the surface (black arrow) and some CG lost their attachment to the plasma membrane (red arrow) to form a secondary layer. (D) NH4Cl-treatment (40 mM pH 9.0, 30 min) shows loss of microvilli and changes in their shape (SEM) as well as translocation of some CG (TEM, red arrows).
	Figure 6. Effects of urethane, procaine and NH4Cl pretreatment on the Ca2+ signaling at fertilization. (A) Ca2+ response in the P. lividus eggs pretreated with 400 mM urethane for 5 minutes, (B) 10 mM procaine for 20 minutes and (C) 40 mM NH4Cl (pH 9.0) for 30 minutes prior to fertilization. The Ca2+ trajectories after fertilization of the same batch of control and treated eggs are presented, and various parameters of the intracellular Ca2+ increase are shown with histograms as average values from three independent experiments. The experimental data are summarized in Table 2.
	Figure 7. Effects of sea urchin egg stratification on microvilli structure and egg surface. (A) Intact sea urchin eggs viewed by SEM in low (left panel) and high magnification (right). Note the microvilli covering the surface. (B) Centrifugation streches the eggs (left panel), corrugates the surface and diminishes the density of microvilli (right panel) (see Table 1). Some of the thickened and elongated microvilli are indicated with arrows. (C) TEM image of a centrifuged egg at the centripetal (left) and centrifugal side (right). While CG are still aligned underneath the plasma membrane in both sides, the cytoplasm at the centripetal and centrifugal sides are predominantly occupied with clear vesicles (CV) and yolk granules (YG), respectively. A minor class of less characterized vesicles enriched with highly electron-desne contents are marked with red arrow. N, nucleus.
	Figure 8. The Ca2+ response in the stratified eggs at fertilization. (A) Pseudo-colored images showing the CF and the beginning of the sperm-initiated Ca2+ wave in intact and centrifuged eggs of P. lividus. Note the secondary calcium increase during the CF (at 5.3 sec) in the centrifugal pole of the centrifuged egg (arrow). Scale bar 20 µm. (B) Comparison of various aspects of the Ca2+ signaling in the intact and stratified eggs. See the text in Results for detail. (C) Calcium curves at fertilization in intact (green) and stratified eggs (brown) from one batch of experiments demonstrating the suppression of the Ca2+ wave in stratified eggs (brown).
	Figure 9. Comparison of the Ca2+ responses at fertilization in the centrifugal and centripetal halves of the stratified eggs. (A) Pseudocolor images of stratified eggs at fertilization showing the sperm's ability to induce Ca2+ increase from both centripetal (CP) and centrifugal (CF) sides of the eggs. (B) Representative Ca2+ curves of the sperm-initiated CFs in the centrifugal (blue line) and centripetal (red line) poles of stratified eggs. The histograms represent the average amplitude of the CF in stratified eggs, which was significantly higher in the centrifugal (0.16 ± 0.06 RFU, n=27) compared to the centripetal pole (0.11 ± 0.05 RFU, n=27; P˂0.001).

References [+] :

Anderson, A cytological study of the centrifuged whole, half, and quarter eggs of the sea urchin, Arbacia punctulata. 1970, Pubmed, Echinobase