Journal: Advanced Science

Article Title: DPAGT1‐Mediated Protein N ‐Glycosylation Is Indispensable for Oocyte and Follicle Development in Mice

doi: 10.1002/advs.202000531

Figure Lengend Snippet: Defective formation of ZP coat and TZPs in oocytes of D166G‐ Dpagt1 mutant females. A) Light micrographs showing the thin and fragile ZP coat in ovulated MUT oocytes. Arrows point to ZP. Scale bars: 50 µm. B) IF staining of ZP1‐3 proteins (green) in GV‐stage immature oocytes in small antral follicles. DNA was counterstained by DAPI (blue). Scale bars indicate 50 µm. C) Transmission electron microscopic graphs of COCs. CC, cumulus cells. Scale bars indicate 5 µm. D) Phalloidin staining of TZPs (red) in immature GV‐oocytes. The left panel is the representative micrographs, and the right graph is the quantification of the number of TZPs per oocyte. Scale bars indicate 50 µm. * p < 0.05, compared to WTs by Student's t ‐test. E) Western blot analysis of ZP1, ZP2, and ACTB expression in GV‐stage immature oocytes.

Article Snippet: Sections were incubated with the primary antibodies to KI‐67 (catalog no. ab16667; 1:200, Abcam, USA), ZP1 (catalog no. sc‐32751; 1:500, Santa Cruz, USA), ZP2 (catalog no. sc‐32752; 1:500, Santa Cruz, USA), and ZP3 (catalog no. sc‐398359; 1:500, Santa Cruz, USA), respectively, followed by Alexa flour 594‐ or 488‐conjugated secondary antibodies (Thermo Fisher Scientific, USA), and were imaged under an LSM 700 confocal laser scanning microscope (Zeiss, Germany).

Techniques: Mutagenesis, Staining, Transmission Assay, Western Blot, Expressing

Journal: Molecular Reproduction and Development

Article Title: Mammalian egg coat modifications and the block to polyspermy

doi: 10.1002/mrd.23320

Figure Lengend Snippet: Scheme of the domain architecture of human and mouse ZP components. The positions of the conserved ZP1‐specific Cys essential for filament cross‐linking (Nishimura et al., ) and the post‐fertilization cleavage site of ZP2 important for ZP hardening (Gahlay, Gauthier, Baibakov, Epifano, & Dean, ) are marked by magenta arrowheads and black arrows, respectively. Dark gray rectangle, SP; light yellow diamond, trefoil/P domain; red rectangle, CFCS; dark yellow rectangle, EHP; brown rectangle, transmembrane domain. The locations of the two ZP3‐specific subdomain elements within the protein's ZP‐C domain, based on the structure of chicken ZP3 (Han et al., ), are indicated by a lighter shading. Experimentally supported (Boja et al., ; Chalabi et al., ; Raj et al., ; Zhao et al., ) and predicted carbohydrates are depicted as solid and dashed inverted tripods, respectively, with N ‐glycans colored black and O ‐glycans colored blue. aa, amino acids, CFCS, consensus furin cleavage site; EHP, external hydrophobic patch; SP, signal peptide; ZP, zona pellucida

Article Snippet: The mouse ZP2 sequence 166 LA|DE 169 , located in the second predicted ZP‐N domain of the protein (Monné et al., ; ZP2 ZP‐N2; Figures and ), corresponds to the experimentally determined cleavage sites in frog gp69/64 (Tian et al., ) and pig ZP2 (Hasegawa, Koyama, Okazaki, Sugimoto, & Isojima, ) and is thus thought to be targeted by murine ovastacin (Burkart et al., ).

Techniques:

Journal: Molecular Reproduction and Development

Article Title: Mammalian egg coat modifications and the block to polyspermy

doi: 10.1002/mrd.23320

Figure Lengend Snippet: Overview of mouse ZP treatments with ZP hardening‐associated factors and the resulting observations

Article Snippet: The mouse ZP2 sequence 166 LA|DE 169 , located in the second predicted ZP‐N domain of the protein (Monné et al., ; ZP2 ZP‐N2; Figures and ), corresponds to the experimentally determined cleavage sites in frog gp69/64 (Tian et al., ) and pig ZP2 (Hasegawa, Koyama, Okazaki, Sugimoto, & Isojima, ) and is thus thought to be targeted by murine ovastacin (Burkart et al., ).

Techniques:

Journal: Molecular Reproduction and Development

Article Title: Mammalian egg coat modifications and the block to polyspermy

doi: 10.1002/mrd.23320

Figure Lengend Snippet: Scheme of the pathway regulating the cleavage of mammalian ZP2. Activation of pro‐ovastacin by a serine protease triggers site‐specific cleavage of ZP2, yielding two protein fragments that remain covalently attached via the predicted C 1 –C 4 disulfide bond of ZP2 ZP‐N2 (indicated by a black bracket). ZP2 cleavage inactivates the sperm‐binding activity of the ZP as well as increases its resistance to α‐chymotrypsin digestion. Premature processing of ZP2 is counteracted by serum glycoprotein fetuin‐B, which inhibits ovastacin and thus, ZP2 cleavage. Structural information is available for ZP2 ZP‐N1 (PDB ID 5II6), ZP2 ZP‐C (PDB ID 5BUP), and fetuin‐B (PDB ID 6HPV). Note that in this figure, as well as in Figure , the ZP module is represented by a single rounded rectangle

Article Snippet: The mouse ZP2 sequence 166 LA|DE 169 , located in the second predicted ZP‐N domain of the protein (Monné et al., ; ZP2 ZP‐N2; Figures and ), corresponds to the experimentally determined cleavage sites in frog gp69/64 (Tian et al., ) and pig ZP2 (Hasegawa, Koyama, Okazaki, Sugimoto, & Isojima, ) and is thus thought to be targeted by murine ovastacin (Burkart et al., ).

Techniques: Activation Assay, Binding Assay, Activity Assay

Journal: Molecular Reproduction and Development

Article Title: Mammalian egg coat modifications and the block to polyspermy

doi: 10.1002/mrd.23320

Figure Lengend Snippet: Possible mechanism of mammalian zona pellucida (ZP) hardening. Whereas ovastacin‐dependent cleavage of the ZP‐N2 domain could induce a conformational change of the ZP2 N‐terminus that leads to new protein–protein interactions, binding of zinc ions to ZP1 ZP‐N1 (based on PDB ID 6GF7) may alter the conformation of ZP filament cross‐links. Together with other ZP modifications discussed in the text, these molecular events could cause tighter interaction between adjacent ZP filaments, resulting in compaction of the supramolecular structure of the egg coat. Modified from (Monné & Jovine, ). Activated ovastacin is indicated by a blue Pac‐Man‐like symbol

Article Snippet: The mouse ZP2 sequence 166 LA|DE 169 , located in the second predicted ZP‐N domain of the protein (Monné et al., ; ZP2 ZP‐N2; Figures and ), corresponds to the experimentally determined cleavage sites in frog gp69/64 (Tian et al., ) and pig ZP2 (Hasegawa, Koyama, Okazaki, Sugimoto, & Isojima, ) and is thus thought to be targeted by murine ovastacin (Burkart et al., ).

Techniques: Protein-Protein interactions, Binding Assay, Modification

Journal: iScience

Article Title: Effects of hyperhomocysteinemia on follicular development and oocytes quality

doi: 10.1016/j.isci.2024.111241

Figure Lengend Snippet: The expression of ZP proteins decreased in female mice with HHcy (A) Schematic representation of ZP production during oocyte growth in mice. (B) Masson staining for changes in the fibrils of follicular ZP at all levels in the ovaries of control and HHcy mice ( n = 3). Arrows indicate the presence of fibrils: yellow arrows indicate the formation of normal ZP fibrils, and black arrows indicate abnormal formation of ZP fibrils. Scale for whole ovary image, 400μm. Scale for follicle images, 25μm. (C) Immunohistochemical detection of ZP2 and ZP3 protein expression in control and HHcy oocytes ( n = 3). Arrow indicates the location of ZP. Scale for primary and secondary follicle images, 50μm. Scale for antral follicle images, 100μm. (D) Western blot probing with anti-ZP2 and anti-ZP3 of oocytes from control and HHcy female mice. (E) Quantitative analysis of ZP2 and ZP3 protein expression in control and HHcy groups. Statistical analysis was performed by Student’s t-test. Data are represented as mean ± SEM ( n = 3). ∗ p < 0.05.

Article Snippet: Rabbit polyclonal antibody to ZP2 , Abmart , Cat#PA5354; RRID: AB_3665051.

Techniques: Expressing, Staining, Control, Immunohistochemical staining, Western Blot

Journal: iScience

Article Title: Effects of hyperhomocysteinemia on follicular development and oocytes quality

doi: 10.1016/j.isci.2024.111241

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal antibody to ZP2 , Abmart , Cat#PA5354; RRID: AB_3665051.

Techniques: Recombinant, Injection, Staining, Software

Journal: JBRA Assisted Reproduction

Article Title: Dietary Soybean ( Glycine max (L.) Merr.) Improved the ZP2 Expression in Female Swiss Mice

doi: 10.5935/1518-0557.20220020

Figure Lengend Snippet: Preparations of mice ovaries with ZP2 marker Immunohistochemical (CPI) staining. Ovaries that were positive (A) and negative (B) for CPI staining with a ZP2 marker, 40× magnification, 100 µm scale bar and primary (C), secondary (D), and tertiary (E and F) follicles that were positive for CPI staining with a ZP2 marker, 400× magnification, 25 µm scale bar.

Article Snippet: Immunohistochemical staining used the IHK marker, namely rabbit zona pellucida 2 (ZP2) antibody (Genetex, GTXGTX64579).

Techniques: Marker, Immunohistochemical staining, Staining

Journal: JBRA Assisted Reproduction

Article Title: Dietary Soybean ( Glycine max (L.) Merr.) Improved the ZP2 Expression in Female Swiss Mice

doi: 10.5935/1518-0557.20220020

Figure Lengend Snippet: The percentage of ZP2 expression in tertiary follicles of mice ovaries for all the study groups. The group with a 50:50 ratio of soybean to pelleted feed (K1), the group with a 25:75 ratio of soybean to pelleted feed (K2), and the control group without soybean administration (K3). The error bar indicates standard deviation (SD). * p <0.05, ** p <0.01, ts=not significant, one-way ANOVA.

Article Snippet: Immunohistochemical staining used the IHK marker, namely rabbit zona pellucida 2 (ZP2) antibody (Genetex, GTXGTX64579).

Techniques: Expressing, Control, Standard Deviation

Journal: Developmental cell

Article Title: Glycan-independent Gamete Recognition Triggers Egg Zinc Sparks and ZP2 Cleavage to Prevent Polyspermy

doi: 10.1016/j.devcel.2018.07.020

Figure Lengend Snippet: (A) Immunoblots of eggs (E) and two-cell embryos (2C) from wildtype and rescue mice augmented with huZP4 to provide a more robust zona pellucida were probed with a monoclonal antibody to ZP2N-term. The number/lane of eggs and embryos from wildtype (left), moZp235−149/huZP4 (middle) and moZp235−262/huZP4 (right) were 10, 100 and 200, respectively. Brackets, moZP2 or moZP2/huZP4 fusion protein. Asterisk, huZP4. Molecular mass, left. Anti-tubulin antibody staining (below) was used as a load control and to ensure protein integrity.

Article Snippet: Membranes were blocked in 5% nonfat milk (BD Biosciences) in Tris-buffered saline (TBS) with 0.05% Tween-20 (Takara Bio USA) and probed with primary antibodies to the N-terminus or C-terminus of ZP2, ZP3 and α-tubulin (MBL) followed by secondary antibodies conjugated to HRP (Jackson ImmunoReserch Laboratories).

Techniques: Western Blot, Staining

Journal: Developmental cell

Article Title: Glycan-independent Gamete Recognition Triggers Egg Zinc Sparks and ZP2 Cleavage to Prevent Polyspermy

doi: 10.1016/j.devcel.2018.07.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Membranes were blocked in 5% nonfat milk (BD Biosciences) in Tris-buffered saline (TBS) with 0.05% Tween-20 (Takara Bio USA) and probed with primary antibodies to the N-terminus or C-terminus of ZP2, ZP3 and α-tubulin (MBL) followed by secondary antibodies conjugated to HRP (Jackson ImmunoReserch Laboratories).

Techniques: Recombinant, Imaging, Expressing, Plasmid Preparation