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Structural and rheological properties conferring fertilization competence to Xenopus egg-coating envelope.
Hanaue M
,
Miwa N
.
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The extracellular egg-coating envelope that comprises a meshwork of filaments polymerized by glycoproteins plays a pivotal role in species-selective sperm recognition and subsequent fertilization; however, the structural and rheological properties conferring fertilization competence to the egg-coating envelope remain poorly unveiled. Here we show several nanoscale-structural and viscoelastic properties of the egg-coat using the transmission electron microscopy and the quartz crystal microbalance experiments, following clamp of the egg-coat at either fertilization-competent or -incompetent statuses by short-term pretreatment with synthetic peptides. Individual filament of approximately 4.8 nm diameter crossed one another, forming several types of intersections. Higher competence-inducing treatment changed the proportion of V-, Y-, and T-type intersections, and induced more randomly deflected angles at intersections. Incompetence-inducing treatment increased the median of a Gaussian distribution of filament lengths that had a peak of 10-20 nm under control conditions; furthermore, this treatment created bumps in the 30-40 and 50-60 nm windows. Quartz crystal microbalance study revealed that viscoelasticity of the competent VE suspension was lower than that of incompetent VE, indicating that viscoelastic property required for successful fertilization resides within a specific range. These findings indicated that the architecture of the egg-coat is capable of rapid and dynamic remodeling, which determines fertilization efficiency.
Figure 1. VE filament meshworks under different fertilization competent statuses. (Upper) Lower magnified TEM images treated with peptides (dcp1 as a control, dcp15 and gpp2; 4 μM; n = 3). Scale bar: 500 nm. (Lower) Higher magnified images. Scale bar: 60 nm.
Figure 2. Distribution of intersection types and deflected angles. (a) Distribution ratio of intersection types, including V-, Y-, T-, X- and Foci types (control, n = 317; +dcp15, VE pretreated with dcp15, n = 224; +gpp2, VE pretreated with gpp2, n = 435). Averaged data were also fitted (mean ± SEM, n = 4). (b) Fitted curves were overlaid (Black, Red and Blue for control, dcp15 and gpp2, respectively). (c) Deflected angles at intersection were measured (control, n = 365; +dcp15, VE pretreated with dcp15, n = 318; +gpp2, VE pretreated with gpp2, n = 677). Averaged data were also fitted (mean ± SEM, n = 4). (d) Fitted curves were overlaid (Black, Red and Blue for control, dcp15 and gpp2, respectively).
Figure 3. Distributions of lengths between intersections. Histograms of lengths between intersections were plotted (control, +dcp15, +gpp2), and averaged data were also fitted (mean ± SEM, n = 4). The histogram for dicalcin-derived peptide-treated VE exhibited bumps at windows of 30–40, 50–60, and 70–80 nm (arrows). Fitted curves were overlaid (Black, Red and Blue for control, dcp15 and gpp2, respectively).
Figure 4. Filament-free area and VE suspension viscoelasticity correlate with fertilization competence. (a) To examine the correlation of filament-free area with fertilization competence, we first measured the ratio of filament-free area against the total area (mean ± SEM, n = 4). (b) The number of intersections per area was measured (mean ± SEM; *P = 0.008, Student’s t-test, n = 4). (c) The ratio of free-space/number of intersection (mean ± SEM; *P = 0.008, Student’s t-test, n = 4). Filament-surrounded free space of fertilization competent VE is significantly smaller than that of fertilization incompetent VE (mean ± SEM; *p = 0.04, Student’s t-test, n = 30), suggesting that fertilization competence is correlated with the filament-surrounded free space. (d) A scheme that represents QCM analysis. Alternating voltage induces the oscillation of the quartz crystal. When a supension of viscous VE is placed on the surface of the quartz crystal, the resonance properties of the quartz oscillation change. (e) A scheme that represents changes in parameters at QCM analysis. Viscous sample evokes an increase in resonance resistance (dR) and a decrease in frequency (df), each of which reflects the viscoelasticity, and the binding to the quartz surface, respectively. (f) Fertilization competence-dependent changes in resonance resistances. Increases in resonance resistance were averaged (mean ± SEM; *P = 0.03, **P = 0.005, Student’s t-test, n = 6–10). Dcp15-treated incompetent VE showed a greater resistance (i.e., greater viscoelasticity) than the control VE, indicating that dicalcin increased the viscoelasticity of the VE. In addition, the FE showed a lower resistance than control, indicating that FE has lower viscoelasticity. Thus, viscoelasticity for successful fertilization resides within a limited range between the iVE and FE values (i.e., 7.5–16 Ω).
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