Fig 1.
AstlmCherry rescues the AstlNull phenotype.
(a) Schematic of the AstlmCherry transgene. (b) Immunoblot of eggs (E) and 2-cell embryos (2C) from wild-type (Ctrl), AstlNull (Null) and AstlNull; AstlmCherry (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), AstlNull; and (e), AstlmCherry 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 AstlmCherry to cortical granules in transgenic mice.
(a) Oocytes (70–75 μm) were isolated from AstlmCherry 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 AstlmCherry 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 AstlmCherry transgenic mice were injected with cRNA encoding UtrCHGFP (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 ovastacinmCherry 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 ovastacinmCherry fusion proteins injected into growing oocytes. Dotted lines indicate the minimal sequence (ovastacinΔ52–64) for localization of the fusion protein to cortical granules. (f) Primary amino acid sequences of ovastacin from 10 mammals were aligned to mouse ovastacin47-69. The conservation of mouse ovastacinΔ52–64 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 ovastacinΔ52–58. (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 AstlΔ/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+/+; AstlmCherry 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 AstlΔ/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 AstlΔ/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 AstlΔ/Δ eggs imaged in (b). N = 30 in each case. (d) In vitro fertilization with wild-type (left), Astl+/Δ (center) and AstlΔ/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 AstlΔ/Δ females co-caged with fertile male mice. The box includes the mean (horizontal line) and data between the 25th and 75th percentile. Error bars indicate the 90th and 10th 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.