Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Mass spectrometric (LC-MS/MS) quantification of ZP protein levels during mouse pre-implantation development. Mouse embryos were collected at consecutive stages of pre-implantation development in vitro after culture in KSOM(aa) medium, and subjected to LC-MS/MS. Zona pellucida was either left in place (zona intact) or removed (zona free). Shown are the relative iBAQ (riBAQ) values of protein abundance for (A) ZP proteins and (B) housekeeping proteins. The color coding, indicative of expression level, was applied in each row independently of the other rows. The raw proteomic data are available in PRIDE repository with accession number PXD056725 and are provided in simplified form in Supplementary Table S1. Abbreviations: riBAQ, relative intensity Based Absolute Quantification [ , ]; LC-MS/MS, Liquid Chromatography with tandem mass spectrometry. Abbreviations: iBAQ, intensity-based absolute quantification; LC-MS/MS, Liquid Chromatography with tandem mass spectrometry; MII, metaphase II oocyte.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, In Vitro, Quantitative Proteomics, Expressing, Liquid Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Immunofluorescence profiling of ZP2 protein during mouse pre-implantation development. (A) Developmental series of embryos cultured in vitro and subjected to indirect IF. (B) Developmental series of embryos cultured in vitro and subjected to direct IF. (C) Developmental series of embryos obtained fresh from the genital tract without in vitro culture and examined by indirect IF. Numbers shown in the top left corner of each diagram indicate the total numbers of embryos examined across all stages. The integrated density values of the ZP2 IF intensities were quantified using Image-J, normalized to the 1-cell stage (set to 1) and plotted by stage of development. Each point corresponds to the normalized integrated density one embryo. Each point corresponds to the normalized integrated density one embryo. All normalized integrated density values are provided in Supplementary Table S2. P-values were calculated with Wilcoxon test. Abbreviations: IF, immunofluorescence; MII, metaphase II (oocyte).

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Immunofluorescence, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Intracellular increase of ZP2 protein abundance is contributed by de novo protein synthesis. (A). Developmental progression of zygotes microinjected with various concentrations of morpholino targeting Zp2 mRNA (treatment) or morpholino targeting an irrelevant sequence (GFP; negative control). Irrespective of the morpholino target, zygotes can hardly progress to the 8-cell stage at morpholino concentrations higher than 0.1 mM. Although this is suggestive of unspecific toxicity of morpholino no matter which target, it should be noted that targeting ZP2 is more detrimental than targeting GFP, as seen from the fact that the green bars in (A) are more extended to the right than the blue bars. At morpholino concentration of 0.05 mM both treatment and control zygotes progressed to blastocyst, however at rates substantially lower than 100% also in the negative control, suggestive of unspecific toxicity. At morpholino concentration of 0.01 mM majority of zygotes progressed to blastocyst in both treatment and control groups, indicating that this concentration is suited to test for gene-specific translation-blocking effects without incurring unspecific toxicity. Numbers of zygotes (n) microinjected with anti-ZP2 morpholino (mM concentration): 34 (0.01), 32 (0.05), 27 (0.1), 24 (0.5), 46 (1.0). Numbers of zygotes microinjected with control morpholino (mM concentration): 36 (0.01), 33 (0.05), 22 (0.1), 22 (0.5), 32 (1.0). (B). Zygotes microinjected with 0.01 mM morpholino were sampled at the 8-cell stage and subjected to ZP2 immunofluorescence using antibody ATCC cat.no. IE-3 – CRL-246. The immunofluorescence intensity values are provided in Supplementary Table S3. Immunofluorescence intensity quantification revealed that ZP2 intensity was reduced by ∼ 20% after translational inhibition of ZP2 mRNA compared to that of negative control. Representative images of the 8-cell embryos are shown after immunostaining with anti ZP2 (ATCC cat.no. IE-3 – CRL-246) revealed by secondary antibody. P-value was calculated with Wilcoxon test. *, <0.05. Abbreviations: IntDen, Integrated density of immunofluorescence signal (intensity x area).

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Quantitative Proteomics, Sequencing, Negative Control, Concentration Assay, Control, Blocking Assay, Immunofluorescence, Inhibition, Immunostaining

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Intracellular ZP2 is required for the morula-to-blastocyst transition and healthy blastocyst formation. Zygotes were microinjected with protein KD reagents and cultured further. (A). At the morula stage (72h post fertilization), ZP2-KD embryos lagged behind in cavitation compared to mock KD and MC embryos. Numbers underneath the box plots are total number of embryos across the stages. The raw data of the developmental rates are provided in Supplementary Table S4. (B) At the blastocyst stage (96h post fertilization), ZP2-KD embryos were deficient in trophectoderm and primitive endoderm compared to mock KD and MC embryos. Numbers underneath the pie charts refer to the blastocysts imaged after triple immunofluorescence. The raw data of the cell counts are provided in Supplementary Table S5. (C) When examined for their ability to develop further, ZP2-KD blastocysts were less able of escaping the zona pellucida and forming outgrowths when plated onto a fibroblast layer in vitro ; in vivo they were less able to develop to term when transferred to pseudopregnant uterus, compared to mock KD and MC blastocysts. Numbers underneath the bar plots are the blastocysts plated on feeders (implantation assay in vitro ) or the fetuses obtained from the blastocysts transferred to uterus (embryo transfer in vivo ). The raw data of the outgrowth and embryo transfers are provided in Supplementary Tables S6 and S7, respectively. P-values were calculated with Wilcoxon test. Abbreviations: ZP2 KD, ZP2 knockdown; mock KD, mock knockdown; MC, micromanipulation control.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Cell Culture, Immunofluorescence, In Vitro, In Vivo, Knockdown, Micromanipulation, Control

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Transcriptomic correlates of unhealthy blastocyst formation after KD of ZP2. Zygotes were microinjected with protein KD reagents and cultured to blastocyst stage. (A) Hierarchical clustering of the 12920 protein-coding mRNAs detected in all samples. (B) Principal component analysis of the samples based on the protein-coding mRNAs. (C) Volcano plots of the mRNAs differentially expressed between ZP2 KD and MC and between mock KD and MC, highlighting the mRNAs that satisfy the criteria of both p<0.05 (Student’s t-test) and |fold change|ti2. The numbers in the colored areas indicate the respective differentially expressed mRNAs. (D) Venn diagram identification of mRNAs differentially exclusively in ZP2 KD and mock KD. Overrepresentation analysis of the mRNAs differentially expressed exclusively in ZP2 KD and mock KD in the ontology “Biological process”. Abbreviations: ZP2 KD, ZP2 knockdown; mock KD, mock knockdown; MC, micromanipulation control.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Cell Culture, Knockdown, Micromanipulation, Control

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Proteomic correlates of unhealthy blastocyst formation after KD of ZP2. Zygotes were microinjected with protein KD reagents and cultured to later morula stage. (A) Hierarchical clustering of the 2401 proteins detected in all samples. (B) Principal component analysis of the samples based on the 2401 proteins. (C) Volcano plots of the proteins differentially expressed between ZP2 KD and MC and between mock KD and MC, highlighting the proteins that satisfy the criteria of both p<0.05 (Student’s t-test) and |fold change|ti2. The numbers in the colored areas indicate the respective differentially expressed proteins. (D) Venn diagram identification of proteins differentially exclusively in ZP2 KD and mock KD and mock. Overrepresentation analysis of the proteins differentially expressed exclusively in ZP2 KD and mock KD in the ontology “Biological process”. Abbreviations: ZP2 KD, ZP2 knockdown; mock KD, mock knockdown; MC, micromanipulation control.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Cell Culture, Knockdown, Micromanipulation, Control

Journal: The Journal of reproduction and development

Article Title: Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization.

doi: 10.1262/jrd.2011-006

Figure Lengend Snippet: Fig. 3. Penetration assays of epididymal sperm through the cumulus. Epididymal sperm from wild-type (WT), Spam1-/- (SPAM1 KO), Spam1-/-/Acr-/- (S/A DKO) and Spam1-/-/Prss21-/- (S/P DKO) mice were labeled with Hoechst 33342, capacitated and mixed with OCCs at a concentration of 150 sperm/μl. After incubation for 15 or 30 min, the cumulus and sperm were fixed with paraformaldehyde. The oocyte ZPs were visualized by immunostaining with anti-ZP2 monoclonal antibody (green colors). Hoechst-labeled sperm (red dots) within the cumulus matrix (CM) and on the oocyte ZP were observed under a microscope (A) and counted (B). Bright-field and fluorescence images were acquired, processed by the MetaMorph software and then merged into one picture. Only pictures obtained 30 min after insemination are shown. A total of 20–24 oocytes were examined for WT, SPAM1 KO, S/A DKO, and S/P DKO sperm (n=4). The differences in the numbers of sperm in the cumulus matrix and on the ZP surface at 15 and 30 min after insemination are significant between WT and SPAM1 KO, S/A DKO or S/P DKO sperm and between SPAM1 KO and S/A or S/P DKO sperm (P<0.001), except between SPAM1 KO and S/A DKO sperm in the cumulus matrix at the 15-min stage (P=0.7). Bar=100 μm.

Article Snippet: Rat monoclonal antibody against mouse ZP2 (IE-3, SC-32752) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Labeling, Concentration Assay, Incubation, Immunostaining, Microscopy, Fluorescence, Software

Journal: PLoS ONE

Article Title: Amyloid Properties of the Mouse Egg Zona Pellucida

doi: 10.1371/journal.pone.0129907

Figure Lengend Snippet: A) Thirty isolated ZP were treated with (+) or without (-) DMSO followed by incubation with the PAD ligand and eluted proteins examined by immunoblot using anti-ZP1, ZP2, and ZP3 antibodies. ZP, an equivalent number of ZP placed directly in SDS-PAGE loading buffer without PAD incubation. Buffer, PBS only incubated with PAD beads. Molecular weight markers indicate kDa. B) phase images of oocytes incubated in the presence (+) or absence (no DMSO) of 90% DMSO for 2 minutes at room temperature. Arrow indicates ZP. Scale bar = 50 μm. C) Equal numbers of ZP were exposed to 90% DMSO, 1% SDS or 0.25% SDS prior to spotting on nitrocellulose using a dot blot apparatus. Membranes were then incubated with the amyloid anti-fibrillar OC and anti-oligomer A11 antibodies. Dot blots were rehybridized with anti-ZP2 antibody to confirm the presence of ZP protein in each well. b, buffer.

Article Snippet: Samples were electro blotted onto a polyvinylidene difluoride membrane (cat. no. IPVH00010, EMD Millipore, Bedford, MA) and the membrane was blocked for 1 h at RT with shaking in 3% nonfat dry milk in TBST and then incubated overnight with shaking at 4°C with either 0.5 μg/ml rat anti-mouse ZP1 (M1.4, sc-32751, Santa Cruz Biotechnology, Santa Cruz, CA), 0.25 μg/ml rat anti-mouse ZP2 (IE-3, sc-32752, Santa Cruz Biotechnology), or 0.5 μg/ml rabbit anti-human ZP3 (H-300, sc-25802, Santa Cruz Biotechnology) in 3% nonfat dry milk in TBST.

Techniques: Isolation, Incubation, Western Blot, SDS Page, Molecular Weight, Dot Blot

Journal: PLoS ONE

Article Title: Amyloid Properties of the Mouse Egg Zona Pellucida

doi: 10.1371/journal.pone.0129907

Figure Lengend Snippet: A) Schematic diagram of mature ZP1, ZP2, and ZP3 with amyloidogenic regions predicted by AmylPred 2 indicated as red bars above the individual domains. Yellow box, trefoil domain. Numbers signify amino acid number. B) Structure based sequence alignment of the ZP polymerization domain of mouse ZP1 (aa 268–541), ZP2 (aa 361–630), and ZP3 (aa 42–305) showing amyloidogenic sites (blue highlighting), as predicted by the Amylpred2 algorithm. Cysteine residues are noted by black boxes. The internal hydrophobic patch (IHP) is indicated by a red box . The β-strand secondary structure based on the crystal structure of chicken ZP3 is noted by orange bars (ZP-N subdomain) and green bars (ZP-C subdomain) above the amino acid sequences . C) Structure based alignment of ZP-N domains in mouse ZP1 (N1, N2), ZP2 (N1-N4), ZP3 (N), abalone VERL repeat 10 (R10) and yeast α-agglutinin/Sag 1p showing predicted amyloidogenic sites in blue highlighting as determined by the AmylPred2 algorithm. The β-strand secondary structure, based on structure of mZP-N is indicated by orange bars above the amino acid sequence . Cysteine residues are noted by black boxes. * , indicate sites essential or important for ZP2-sperm and α-agglutinin-a-agglutinin binding [ , ].

Article Snippet: Samples were electro blotted onto a polyvinylidene difluoride membrane (cat. no. IPVH00010, EMD Millipore, Bedford, MA) and the membrane was blocked for 1 h at RT with shaking in 3% nonfat dry milk in TBST and then incubated overnight with shaking at 4°C with either 0.5 μg/ml rat anti-mouse ZP1 (M1.4, sc-32751, Santa Cruz Biotechnology, Santa Cruz, CA), 0.25 μg/ml rat anti-mouse ZP2 (IE-3, sc-32752, Santa Cruz Biotechnology), or 0.5 μg/ml rabbit anti-human ZP3 (H-300, sc-25802, Santa Cruz Biotechnology) in 3% nonfat dry milk in TBST.

Techniques: Sequencing, Binding Assay