Journal: bioRxiv
Article Title: A mechanical origin for implantation defects in embryos from aged females
doi: 10.1101/2025.09.29.679218
Figure Lengend Snippet: a) Immunostaining of late E4.5 blastocysts with the tension sensor, VD7. The VD7 antibody is an engineered antibody sequence based on vinculin’s binding association to alpha-catenin. Contractility at cell-cell junctions creates a force-dependent conformational change in alpha-catenin proteins that exposes this epitope pocket to which vinculin binds. VD7 association is observed when E-cadherin-mediated adhesions experience tension, making it an indicator of tissue under tensile strain. We measured global junctional VD7 and normalized its levels to total alpha catenin to quantify relative junctional VD7 signal intensity. Blastocyst fixed and stained for tension sensor VD7, showing increasing junctional VD7 towards the mural trophectoderm region. Normalized VD7 (purple) was calculated over total alpha-catenin (black), showing that maternally aged embryos show higher junctional tensions both at the mural and polar trophectoderm regions. (Young N = 4, n = 386 pTE junctions, 532 mTE junctions, 10 blastocysts; Aged N = 3, n = 218 pTE junctions, 383 mTE junctions, 8 blastocysts). p values from Mann-Whitney test. Scale bar is 10μm. b) Plot showing Normalized junctional NMIIB staining in the mTE region, with higher junctional accumulation in the aged condition (left) (Young N = 2, n = 1,610 mTE junctions, 17 blastocysts; Aged N = 2, n = 935 mTE junctions, 14 blastocysts). p values from Mann-Whitney test. Scale bar is 10μm. Representative images of NMIIB staining (purple) in both maternal conditions. Scale bar is 5μm and 10μm. c) Plot showing Normalized junction pMLC staining in the mTE region, with higher junctional accumulation in the aged condition (Young N = 2, n = 1,610 mTE junctions, 17 blastocysts; Aged N = 2, n = 935 mTE junctions, 14 blastocysts). p values from Mann-Whitney test.
Article Snippet: Primary antibodies used were: Rabbit pMLC (1:200; Cell Signaling Cat#3671S), Mouse ZO-1 clone 1A12 (1:200; Invitrogen Cat#33-9100), Goat ZO-1 (1:200; Invitrogen Cat#PA5-19090), Rabbit VD7 (2mg/ml, a kind gift from Dietmar Vestweber lab), Rabbit ppMLC (1:200; Cell Signaling Cat#3674T), Mouse Alpha-catenin clone 7A4 (1:500; Thermo Fisher Cat#13-9700), Rabbit NMIIA (1:200, BioLegend Cat#909802) Rat NMIIB (Abcam Cat# (EPR22564-23), Rabbit NMIIB (BioLegend Cat#909902).
Techniques: Immunostaining, Sequencing, Binding Assay, Staining, MANN-WHITNEY
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