Limatola N et al. (2021), Effects of Dithiothreitol on Fertilization and ...

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Cells 2021 Dec 17;1012:. doi: 10.3390/cells10123573.

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Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin.

Limatola N , Chun JT , Cherraben S , Schmitt JL , Lehn JM , Santella L .

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The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg's fertilization response.

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	Figure 1. Ultrastructure of unfertilized sea urchin eggs at the surface after brief treatment with dithiothreitol. P. lividus eggs were exposed to 10 mM dithiothreitol (DTT) in seawater for 10 min and were subjected to analyses by electron microscopy, following the procedures described in Materials and Methods. (A,B) Images obtained by SEM. (C,D) Images obtained by TEM. Note that, after the DTT treatment, the shape of the individual microvilli (MV) became irregular and slightly thicker, and that numerous blebs were formed on the egg surface (yellow and red arrows in B,D,F). (E,F) For closer examination, the areas of the TEM images marked by brackets in panels (C,D) are enlarged. Scale bars, 1 μm. CG—cortical granules; MV—microvilli; M—mitochondria; Y—yolk granules; VL—vitelline layer.
	Figure 2. Ultrastructure of the egg surface immediately after fertilization. P. lividus eggs were fertilized and fixed for SEM (A,B,D,E) and TEM (C,F) analyses. (A–C) Control eggs fertilized in NSW. (D–F) Eggs preincubated (10 min) and fertilized in seawater containing 10 mM DTT. Images in (A,D) were captured by fixing the eggs 25 s after insemination. The images in all the other panels show the ultrastructure of the egg surface 1 min after insemination. Note that, in panel (E), most parts of a fertilizing sperm were not internalized yet, but were surrounded by the egg protrusion through the ruptured fertilization envelope (red arrow). Scale bars: panels (B,E), 10 μm; all other panels, 1 μm. FE, fertilization envelope.
	Figure 3. Ultrastructure of the egg surface at a later stage of fertilization. P. lividus eggs were fertilized and fixed 5 min later for SEM and TEM analyses. (A,C,E) Control eggs fertilized in NSW. (B,D,F) Eggs preincubated (10 min) and fertilized in seawater containing 10 mM DTT. Panels (A−D) show SEM images, while panels (E,F) represent results of TEM. Red arrows in panels (D,F) indicate the ruptured fertilization envelope. A blue arrowhead in panel F indicates the over-elongated microvilli. Scale bars: panels (A,B), 10 μm; all other panels, 1 μm. FE, fertilization envelope (arrows); HL, hyaline layer.
	Figure 4. DTT pretreatment of the eggs alters the Ca2+ response at fertilization. P. lividus eggs were microinjected with calcium dye and preincubated in seawater with 10 mM DTT (10 min) prior to fertilization. As a control (CTL), eggs from the same batch were prepared in parallel and fertilized in NSW. For comparison, some eggs preincubated in the presence of 10 mM DTT were restored in normal seawater and fertilized (WASH). (A) Instantaneous increment of Ca2+ signals inside a control egg visualized in pseudocolour. The time point immediately before the first detectable Ca2+ signal was set as t = 0. The arrow indicates cortical flash (CF), and the arrowhead pinpoints the sperm interaction site on the egg where the Ca2+ wave is initiated. (B) Changes of the Ca2+ signals: green curves (CTL), brown curves (eggs fertilized in 10 mM DTT), and curves in pale blue (eggs fertilized after restoration in NSW, 10 min). The vertical arrow indicates the CF. Out of four independent experiments conducted, the result of one representative experiment was plotted for Ca2+ trajectories. Histograms in panels C−F represent comparisons of the egg groups with respect to the CF amplitude (C), the peak amplitude of the Ca2+ wave (D), the duration of the Ca2+ wave traversing the egg from the sperm interaction site to the antipode (E), and the time interval between the CF and the initiation of the Ca2+ wave (F). * Tukey’s post hoc test, p < 0.01. RFU, relative fluorescence unit.
	Figure 5. F-actin mobilization during egg activation is inhibited by DTT, the membrane-permeant reducing agent. P. lividus eggs were microinjected with F-actin dye, AlexaFluor 568-phalloidin, and preincubated in seawater in the presence or absence (control) of 10 mM DTT for 10 min prior to fertilization. For comparison, some of the control eggs were exposed to another type of reducing agent that does not penetrate cell membrane, tris(2-carboxyethyl)phosphine (TCEP). The changes of the actin cytoskeleton following fertilization were monitored by confocal microscopy at intervals. The moment of insemination was set as t = 0. The white dotted curves drawn on the images at 30 min delineate the borders of the egg surface. To visualize the fertilization envelope, the same AlexaFluor 568-phalloidin eggs were shown in the bright field view 40 min after insemination. These images are representative of at least two independent experiments. Scale bar, 10 μm.
	Figure 6. Effect of DTT pretreatment on the late events of fertilization. (A–C) P. lividus eggs were microinjected with F-actin dye, AlexaFluor 568-phalloidin, and preincubated in seawater in the presence or absence (control) of 10 mM DTT for 10 min prior to fertilization. After 10 min, the actin cytoskeleton of the fertilized eggs was visualized by confocal microscopy. The fertilization cones are marked by arrowheads. (D–I) Eggs pretreated in the same conditions as above (but without F-actin dyes) were fertilized and incubated in the presence of FM 1-43 (D–F) or BPA-C8-Cy3 (G–I). White arrows indicate the fertilization envelope and its remnants. (J–L) Hoechst 33,342 introduced with sperm visualized the male and female pronuclei of the zygotes in the given conditions, as indicated by pale blue and pink arrows, respectively. The black arrow indicates the fertilization envelope. These images are representative of at least two independent experiments. Scale bar, 10 μm.
	Figure 7. DTT pretreatment of the eggs induces anomalies during early development. (A–D) P. lividus eggs were incubated in the presence or absence (control) of 10 mM DTT for 10 min and fertilized. (E–F) Some of the DTT-pretreated eggs were rinsed and restored in NSW (10 min) and fertilized (WASH). The cleavage and development of the embryos in each condition were monitored by light microscopy. Arrowheads indicate the FE. Note that the FE covering the control embryo is still intact at 6 h (B), where it is totally absent in the embryos fertilized in the presence of DTT. The FE is evident again in the embryos derived from the eggs restored in NSW prior to fertilization (F). Red arrows indicate signs of leakage in the embryo from the ruptured FE. Scale bar, 10 μm. FE, fertilization envelope. The number of fertilized eggs examined for each set of experiments was 100. Representative results from one of the three independent experiments are shown.

References [+] :

Aketa, On the sperm-egg bonding as the initial step of fertilization in the sea urchin. 1967, Pubmed, Echinobase