A Unique Egg Cortical Granule Localization Motif Is Required for Ovastacin Sequestration to Prevent Premature ZP2 Cleavage and Ensure Female Fertility in Mice

Abstract

Monospermic fertilization is mediated by the extracellular zona pellucida composed of ZP1, ZP2 and ZP3. Sperm bind to the N-terminus of ZP2 which is cleaved after fertilization by ovastacin (encoded by Astl) exocytosed from egg cortical granules to prevent sperm binding. AstlNull mice lack the post-fertilization block to sperm binding and the ability to rescue this phenotype with AstlmCherry transgenic mice confirms the role of ovastacin in providing a definitive block to polyspermy. During oogenesis, endogenous ovastacin traffics through the endomembrane system prior to storage in peripherally located cortical granules. Deletion mutants of ovastacinmCherry expressed in growing oocytes define a unique 7 amino acid motif near its N-terminus that is necessary and sufficient for cortical granule localization. Deletion of the 7 amino acids by CRISPR/Cas9 at the endogenous locus (AstlΔ) prevents cortical granule localization of ovastacin. The misdirected enzyme is present within the endomembrane system and ZP2 is prematurely cleaved. Sperm bind poorly to the zona pellucida of AstlΔ/Δ mice with partially cleaved ZP2 and female mice are sub-fertile.

Fig 1. Astl^mCherry rescues the Astl^Null phenotype.

(a) Schematic of the Astl^mCherry transgene. (b) Immunoblot of eggs (E) and 2-cell embryos (2C) from wild-type (Ctrl), Astl^Null (Null) and Astl^Null; Astl^mCherry (Rescue) mice (10–15 eggs or embryos per lane). Primary antibody (M2c.2) binds to the C-terminus of ZP2 and detected intact (upper arrow) and cleaved (lower arrow) protein. Molecular mass (kD) on left. (c-e) Mouse sperm binding to eggs and two-cell embryos from: (c), wild-type; (d), Astl^Null; and (e), Astl^mCherry rescue mice. Confocal projections (upper) and DIC (lower) images were obtained after fixation and staining with Hoechst. Scale bar, 20 μm.

Fig 2. Localization of Astl^mCherry to cortical granules in transgenic mice.

(a) Oocytes (70–75 μm) were isolated from Astl^mCherry transgenic mice and imaged in an environmental chamber (37°C, 5% CO₂) by confocal microscopy during germinal vesicle (nucleus) breakdown (GVBD). (b) GV-intact oocytes (upper), ovulated metaphase II (MII) eggs (middle) and one cell (1C) embryos (lower) from Astl^mCherry transgenic mice were permeabilized, stained with antibody to ovastacin and with LCA-FITC to localize cortical granules and imaged with confocal and DIC microscopy. CG, cortical granule; PB, polar body. Scale bar, 20 μm. (c) GV-intact oocytes from Astl^mCherry transgenic mice were injected with cRNA encoding UtrCH^GFP (calponin homology domain of utrophin) to localize the actin cap associated with formation of the cortical granule free region during meiotic maturation. Live-images were obtained over time (4 hr) using confocal and DIC microscopy. Scale bars, 20 μm.

Fig 3. Ovastacin cortical granule localization motif.

(a) Growing oocytes (60–65 μm) were injected with cRNA encoding an ovastacin^mCherry fusion protein (top) with a signal peptide (1–23 aa), a pro segment (24–85 aa) and the mature ovastacin enzyme (86–435 aa) containing an active site (asterisk, ¹⁸²HELMHVLGFWH¹⁹²) fused to mCherry (236 aa). Six hr after injection, oocytes were fixed, permeabilized and stained with antibodies to ovastacin, LCA-FITC and Hoechst and imaged by confocal and DIC microscopy (bottom). Scale bar, 20 μm. (b) Same as (a) after injection of cRNA encoding either ovastacin 1–89 aa or ovastacin 1–23; 82–435 aa indicated in (e). (c) Same as (a) after injection of cRNA encoding either ovastacin 1–64 aa or ovastacin 1–23; 61–435 aa indicated in (e). (d) Same as (a) after injection of cRNA encoding either ovastacin 1–51 aa or ovastacin 1–28; 52–64 aa indicated in (e). (e) Schematic representation of three pairs of complementary deletion constructs of ovastacin^mCherry fusion proteins injected into growing oocytes. Dotted lines indicate the minimal sequence (ovastacin^Δ52–64) for localization of the fusion protein to cortical granules. (f) Primary amino acid sequences of ovastacin from 10 mammals were aligned to mouse ovastacin^47-69. The conservation of mouse ovastacin^Δ52–64 cortical granule localization signal is indicated by asterisks. (g) Same as (a) after injection of cRNA encoding ovastacin 1–28; 52–58.

Fig 4. Ovastacin is not present in cortical granules after partial deletion of the localization motif.

(a) Schematic representation of the CRISPR/Cas9 deletion mutation in Astl exon 2 that encodes ovastacin^Δ52–58. (b) Wild-type GV-intact oocytes were fixed, permeabilized and stained with antibodies to ovastacin, LCA-FITC and imaged by confocal and DIC microscopy. (c) Same as (b) with GV-intact oocytes, ovulated MII eggs, 1-cell zygotes and 2-cell embryos from Astl^Δ/Δ mice. (d) Same as (b) with GV-intact oocytes and ovulated MII eggs from Astl^+/Δ mice. (e) GV-intact oocytes and ovulated MII eggs from Astl^+/+; Astl^mCherry and Astl^+/Δ; Astl ^mCherry mice were fixed, permeabilized and imaged by confocal and DIC microscopy. Scale bars, 20 μm.

Fig 5. Premature cleavage of ZP2 affects fertility of Astl mutant mice.

(a) Immunoblot of zonae pellucidae from wild-type (Ctrl), Astl^+/Δ and Astl^Δ/Δ ovulated eggs as well as wild-type 2C embryos (Ctrl) stained with a monoclonal antibody (M2c.2) that binds to the C-terminus of ZP2 and detected intact (upper arrow) and cleaved (lower arrow) protein. Molecular mass (kD) on left. (b) Sperm binding to wild-type eggs, 2C embryos, Astl^+/Δ and Astl^Δ/Δ eggs. Confocal projections (upper) and DIC (lower) images were obtained after fixation and staining nuclei with Hoechst. Arrows, nuclei; PB, polar body. Scale bar, 20 μm. (c) Average (± s.e.m) number of sperm bound to wild-type eggs (Ctrl), 2C embryos (Ctrl), Astl^+/Δ and Astl^Δ/Δ eggs imaged in (b). N = 30 in each case. (d) In vitro fertilization with wild-type (left), Astl^+/Δ (center) and Astl^Δ/Δ (right) eggs with and without zonae pellucidae. Fertilization was determined by the presence of 2 pronuclei 12 hr after insemination. (e) Dot density (left) and associated box plots (right) of litters from wild-type (Control), Astl^+/Δ and Astl^Δ/Δ females co-caged with fertile male mice. The box includes the mean (horizontal line) and data between the 25^th and 75^th percentile. Error bars indicate the 90^th and 10^th percentiles and outliers are indicated by dots. Statistical differences in c and d were determined by 2-tailed Student’s T-test, P <0.001.