Wozniak KL et al. (2018), PLC and IP3-evoked Ca2+ release initiate the fa...

XB-ART-55112

J Gen Physiol 2018 Sep 03;1509:1239-1248. doi: 10.1085/jgp.201812069.

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PLC and IP3-evoked Ca2+ release initiate the fast block to polyspermy in Xenopus laevis eggs.

Wozniak KL, Tembo M, Phelps WA, Lee MT, Carlson AE.

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The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg's membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca2+ in the African clawed frog, Xenopus laevis, which in turn activates an efflux of Cl- that depolarizes the cell. Here we seek to identify the source of this intracellular Ca2+ Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca2+ entry into the egg from the extracellular milieu and Ca2+ release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca2+-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C-a potent inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum (ER)-completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP3-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases IP3 and evokes Ca2+ release from the ER. This ER-derived Ca2+ then activates a Cl- channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.

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Species referenced: Xenopus laevis
Genes referenced: ano1 cacna1d cacng6 dennd5a dennd5b itpr1 mcu nadsyn1 ncs1 orai2 pkd2 plcb1 slc24a3 slc24a5 tmco1 tmem37 tpcn2 trpc3 trpv2 trpv4
GO keywords: prevention of polyspermy

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	Figure 1. Fertilization signals a depolarization in X. laevis eggs. (A) Representative whole-cell recordings made during fertilization in control conditions (in MR/5 solution). Dashed line denotes 0 mV. (B) Images of X. laevis (top) egg before sperm addition and (bottom) egg ∼15 min after fertilization with animal pole contracted. Tukey box plot distributions of the (C) resting and fertilization potentials in control conditions and (D) depolarization rate (n = 31, recorded over 22 experiment days). The central line represents the median value, the box denotes the data spread from 25 and 75%, and the whiskers reflect 10–90%.
	Figure 2. Expression of Ca2+ channels in X. laevis eggs. Heatmaps of RNA (left) and protein (Prot; right) expression levels of Ca2+ channels whose transcript levels were >1 TPM. Transcript levels (shown as TPM) were obtained from Session et al. (2016). Protein concentrations are from Wühr et al. (2014) as determined by mass spectrometry (mass spec.)–based proteomics in log2 nanomolar. Red arrows highlight plasma membrane localized Ca2+ channels found in eggs.
	Figure 3. Fertilization-signaled depolarization does not require Ca2+ entry into X. laevis eggs. (A and B) Representative fertilization recordings made in solutions with 10 µM GdCl3 (A) and 20 µM SK&F-96365 (B). Dashed lines denote 0 mV. (C–E) Tukey box plot distributions of the resting (C) and fertilization (D) potentials and depolarization rate (E) for indicated treatments (n = 8–12, recorded over 3 experiment days per treatment). In D and E, the gray lines denote the Tukey box plot distributions for recordings made in control conditions (in the MR/5 solution), where the solid line represents the median value, the dashed lines denote the data spread from 25 and 75%, and the whiskers reflect 10–90%. (F) Images of X. laevis embryos from monospermic (top) and polyspermic (bottom) fertilizations. (G) Proportion of polyspermic embryos out of total developed embryos in control, Gd3+, and SK&F-96365 (n = 3–6, recorded over 3–6 experiment days per treatment, the mean values ± SEM are reported).
	Figure 4. ER-released Ca2+ is essential for the fast polyspermy block. Representative fertilization recordings made in the presence of 500 nM Xestospongin C (XC; A), vehicle (2% DMSO; C), or 100 µM 2-APB (F). Dashed line denotes 0 mV, and arrows indicate sperm additions. Tukey box plot distributions of the resting (B) and fertilization (D) potentials, as well as the depolarization rates (E), from recordings made in the indicated treatments (n = 5–8, 2–4 experiment days per treatment). In B and D, the gray lines behind the box plots represent the Tukey box plot distributions for the control (MR/5) data, where the solid line represents the median value, the dashed lines denote the data spread from 25 and 75%, and the whiskers reflect 10–90%. (G) Proportion of polyspermic embryos out of total developed embryos in vehicle (2% DMSO in MR/5), Xestospongin C, control (MR/5), and 2-APB (n = 3, recorded over 3 experiment days per treatment, the mean values ± SEM are reported). *, P < 0.05.
	Figure 5. PLC is required for the fast block. (A and B) Representative fertilization recordings made in 1 µM U73122 (A) or 1 µM U73343 (B). Dashed line denotes 0 mV, and arrows indicate sperm additions. (C–E) Tukey box plot distributions of the resting (C) and fertilization (D) potentials and the depolarization rates (E) made in indicated treatments (n = 7–8, recorded over 3–5 experiment days per treatment, the mean values ± SEM are reported). The gray lines behind the box plots represent the Tukey box plot distributions for the control (MR/5) data, where the solid line represents the median value and the dashed lines denote the data spread from 25 and 75% and the whiskers reflect 10–90%. (F) Percent polyspermic embryos out of total developed embryos in control (MR/5), U73122, and U73343. , P < 0.05; *, P < 0.01.
	Figure 6. Model for the fast polyspermy block in X. laevis. In the fast block to polyspermy in X. laevis eggs, fertilization activates a PLC, which then cleaves PIP2 to create IP3. This increased IP3 then activates its cognate receptor on the ER to evoke a Ca2+ release. This ER-derived Ca2+ then activates a Cl− channel, which conducts a Cl− efflux to depolarize the egg. Research described in the companion paper (Wozniak et al., 2018) identifies this Cl− channel as TMEM16A. This fertilization-signaled depolarization prevents sperm entry into an already-fertilized egg.

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Berridge, Inositol trisphosphate and calcium signalling mechanisms. 2009, Pubmed