Journal: Advanced Science

Article Title: Granulosa Cell‐Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post‐Ovulatory Follicle

doi: 10.1002/advs.202403640

Figure Lengend Snippet: Spatial transcriptome analysis suggested that mGC‐layer stiffening may result from the assembly of focal adhesions. A) Identification of pre‐ovulatory follicles within the ovaries through spatial transcriptome analysis. n = 7 (H0) and 10 (H6) pre‐ovulatory follicles, respectively. The mGCs within the pre‐ovulatory follicles are highlighted in blue. B) PCA analysis of the transcriptome differences in mGCs within pre‐ovulatory follicles. C) Heat map of the up‐regulated genes in mGCs after hCG injection. D) GO analysis of the up‐regulated genes. Biological processes related to cell adhesion are outlined by yellow frames. E) Heat map of genes encoding the components of tight junction, desmosome, hemidesmosome, and focal adhesion. F) qRT‐PCR validation of the expression of genes encoding the components of tight junction, desmosome, hemidesmosome, and focal adhesion. n = 6 mGC samples. G) Schematic representation of the structure of a focal adhesion. H) Western blot assay of the protein contents of VCL and TLN1 after hCG injection. n = 3 mGC samples. Original blots can be viewed in Figure (Supporting Information). I) Immunofluorescence analysis of the localization of VCL and TLN1 in follicle after hCG injection. Nuclei (blue) were stained with DAPI. J) qRT‐PCR analysis of the expression of goat genes encoding the components of focal adhesion after LH injection. L0: 0 h post LH injection, L10: 10 h post LH injection. n = 5 (L0) and 4 (L10) mGC samples. K) Analysis of the transcriptome changes in monkey mGC after hCG injection. Left : Heat map of the up‐regulated genes; right : GO analysis of the up‐regulated genes. Focal adhesion outlined by yellow. L) Analysis of the transcriptome changes in human mGC after hCG injection. Left : Heat map of the up‐regulated genes; right : GO analysis of the up‐regulated genes. Focal adhesion outlined by yellow. Statistical significance was determined using two‐tailed unpaired Student's t‐test, values were mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001. F, H, and I were repeated independently three times, yielding consistent results.

Article Snippet: The same protocol was applied for TLN1 detection using the primary antibody against TLN1 (1:300 dilution, 14168‐1‐AP, Proteintech, USA) and fluorescent dye TYR‐690 (Recordbio Biological Technology, China) for TLN1 staining.

Techniques: Injection, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Granulosa Cell‐Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post‐Ovulatory Follicle

doi: 10.1002/advs.202403640

Figure Lengend Snippet: Disruption of focal adhesion assembly led to a failure in the stiffening of mGC‐layer and an escape of mGCs from the punctured follicle. A) Schematic representation of the knockdown of VCL and TLN1 in cultured follicles. B) Effect of VCL and TLN1 knockdown on follicle growth, n = 3 ( si‐Control ), 4 ( si‐VCL ); 4 ( si‐Control ), 4 ( si‐TLN1 ); 18 ( si‐Control ), 11 ( si‐VCL+TLN1 ). The scrambled shRNA was used as si‐Control in this study. C) Effect of VCL and TLN1 knockdown on the capability of mGC‐layer to escape from the punctured follicles. The mGC‐layers are outlined by yellow frames. D) Effect of VCL and TLN1 knockdown on the rigidity of mGC‐layer. Oscillation parameters: 700 rpm, 1 min, and 37 °C. E) Schematic representation of the knockdown of VCL and TLN1 in ovaries. F) Efficiency analysis of VCL + TLN1 interference using qRT‐PCR and western blot, n = 3 ovaries, collected from 3 mice. Green fluorescence indicates successful transcription of interfering plasmids in ovaries. Original blots can be viewed in Figure (Supporting Information). G) Effect of VCL + TLN1 knockdown on the capability of mGC‐layer to escape from punctured ovaries. mGC‐layers outlined by yellow frames. H) Effect of VCL + TLN1 knockdown on the rigidity of mGC‐layer. Oscillation parameter: 700 rpm, 1 min, and 37 °C. Statistical significance was determined using two‐tailed unpaired Student's t test, with values presented as mean ± SD. **** p < 0.0001. C, D were repeated independently five times, and G, H were repeated two times, yielding consistent results.

Article Snippet: The same protocol was applied for TLN1 detection using the primary antibody against TLN1 (1:300 dilution, 14168‐1‐AP, Proteintech, USA) and fluorescent dye TYR‐690 (Recordbio Biological Technology, China) for TLN1 staining.

Techniques: Disruption, Knockdown, Cell Culture, Control, shRNA, Quantitative RT-PCR, Western Blot, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Granulosa Cell‐Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post‐Ovulatory Follicle

doi: 10.1002/advs.202403640

Figure Lengend Snippet: Disruption of focal adhesion assembly resulted in the release of mGCs from the post‐ovulatory follicle and a reduction in the quantity of luteal cells. A) Schematic representation for real‐time recording of ovulation process after VCL+TLN1 knockdown. B) Effect of VCL+TLN1 knockdown on the ovulation rate. C) Knocking down VCL+TLN1 results in the spontaneous release of mGCs from the post‐ovulatory follicle. Left : Representative photographs of the post‐ovulatory follicles. The free mGCs released from the rupture site are outlined by red frames; the mGC clumps protruded from the rupture site are outlined by yellow frames; the released COCs are covered by green frames. Right : identity verification of the released mGCs through qRT‐PCR. n = 4 cellular samples. Lhcgr was chosen as the marker gene for mGC. Purified mGCs and cumulus cells were used as positive and negative controls, respectively. D) Effect of VCL+TLN1 knockdown on the morphology and function of the in vitro corpus luteum. Left : representative photographs of luteal sections in each group. Right : statistics of the density of luteal cells in each group, n = 7 ( si‐Control ), 8 ( si‐VCL+TLN1 ). E) Effect of VCL+TLN1 knockdown on progesterone level in culture medium. n = 10 medium samples in each group. F) Experimental design of G , H . G) Effect of VCL+TLN1 knockdown on the morphology and function of the in vivo corpus luteum. Left : representative photographs of ovarian sections. CL = corpus luteum, which are outlined by yellow frames. The CLs with cavities are outlined by green frames; F = follicle. Scale bar in the enlarged image: 100 µm. Right : statistics of the density of luteal cells, n = 22 ( si‐Control ), 21 ( si‐VCL+TLN1 ) CLs. These CLs were observed from 4 and 7 biological independent ovaries, respectively. H) Effect of VCL+TLN1 knockdown on progesterone level in serum. n = 7 serum samples in each group. Statistical significance was determined using two‐tailed unpaired Student's t test and Chi‐squared test, values were mean ± SD. *** p < 0.001, **** p < 0.0001. C and D were repeated independently five times, G was repeated two times, yielding consistent results.

Article Snippet: The same protocol was applied for TLN1 detection using the primary antibody against TLN1 (1:300 dilution, 14168‐1‐AP, Proteintech, USA) and fluorescent dye TYR‐690 (Recordbio Biological Technology, China) for TLN1 staining.

Techniques: Disruption, Knockdown, Quantitative RT-PCR, Marker, Purification, In Vitro, Control, In Vivo, Two Tailed Test

Journal: Advanced Science

Article Title: Granulosa Cell‐Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post‐Ovulatory Follicle

doi: 10.1002/advs.202403640

Figure Lengend Snippet: Ovulatory signal stimulated focal adhesion assembly and mGC‐layer stiffening by activating the cAMP‐PKA‐CREB cascade. A) KEGG analysis of the up‐regulated genes. B) Analysis of the binding of CREB to promoter of focal adhesion structural genes. The binding peaks of CREB are indicated by red triangles. C) Western blot assay of the protein contents of VCL and TLN1 following activation or inhibition of the cAMP‐PKA cascade. Original blots can be viewed in Figure (Supporting Information). D) Effect of activating or inhibiting cAMP‐PKA cascade on the rigidity and escape capability of the mGC‐layer. The released mGC‐layers are outlined by yellow frames. E) ChIP‐qPCR assay for CREB binding to the promoters of VCL and TLN1 . Up : electrophoretic images of PCR products. Input and IgG were used as positive and negative controls, respectively. Original gel images can be viewed in Figure (Supporting Information). Down : statistical chart of qPCR assay. n = 4 mGC samples. F) Effect of CREB knockdown on focal adhesion assembly and “m GC‐layer stiffening”. Left : western blot assay of protein contents of VCL and TLN1 after CREB knockdown. Original blots can be viewed in Figure (Supporting Information). Right : Effect of CREB knockdown on the rigidity and escape capability of the mGC‐layer. The released mGC‐layers are outlined by yellow frames. Statistical significance was one‐way ANOVA followed by Tukey's post hoc test, values were mean ± SD. * p < 0.05, ** p < 0.01. C, D, and F were repeated independently three times, E was repeated two times, yielding consistent results.

Article Snippet: The same protocol was applied for TLN1 detection using the primary antibody against TLN1 (1:300 dilution, 14168‐1‐AP, Proteintech, USA) and fluorescent dye TYR‐690 (Recordbio Biological Technology, China) for TLN1 staining.

Techniques: Binding Assay, Western Blot, Activation Assay, Inhibition, ChIP-qPCR, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: Podocyte histone deacetylase activity regulates murine and human glomerular diseases

doi: 10.1172/JCI124030

Figure Lengend Snippet: (A) Representative glomerular immunofluorescence images of talin-1 (green) and nephrin (red) in doxycycline-induced (Dox-induced) control and Tln1fl/fl Pod-rtTA TetO-Cre mice. Scale bar: 10 μm. (B) Quantification of urine albumin/creatinine ratio in control and Tln1fl/fl Pod-rtTA TetO-Cre mice at 0, 2, and 4 weeks after completion of Dox induction. *P < 0.05 vs. control; n = 5. (C) Plasma creatinine (Cr) levels in control and Tln1fl/fl Pod-rtTA TetO-Cre mice treated with Dox at 0 and 4 weeks after completion of Dox induction. *P < 0.05 vs. control; n = 5. (D) Representative light microscope images (H&E, periodic acid–Schiff [PAS], and trichrome) of Dox-induced control and Tln1fl/fl Pod-rtTA TetO-Cre mouse glomeruli. Arrowheads show mesangial matrix deposition and mesangial cell proliferation. Scale bar: 25 μm. (E) Representative trichrome staining in control and Tln1fl/fl Pod-rtTA TetO-Cre mouse kidneys at representative time points. Arrowheads depict dilated tubules and proteinaceous casts, and arrows display interstitial fibrosis. Scale bar: 50 μm. (F) Representative transmission electron micrograph (TEM) in control and Tln1fl/fl Pod-rtTA TetO-Cre mouse foot processes after Dox induction. Arrowheads depict podocyte foot process effacement. Scale bar: 1 μm. (G) Quantification of glomerulosclerosis in D. *P < 0.05 vs. control. (H) Quantification of interstitial fibrosis in E. *P < 0.05 vs. control. (I) Quantification of foot processes in F. *P < 0.05 vs. control. (J) Quantification of WT1-positive number per glomerulus in control and Tln1fl/fl Pod-rtTA TetO-Cre mice treated with Dox at 0 and 4 weeks after completion of Dox induction. *P < 0.05 vs. control; n = 3. (B, C, and G–J) Statistically analyzed by 1-way ANOVA with Dunnett’s correction.

Article Snippet: Mouse anti–Wilms tumor 1 (anti-WT1) (EMD Millipore Corp., catalog 05-753); rabbit anti-WT1 (Santa Cruz Biotechnology, catalog sc-192); mouse anti-HDAC1 (catalog 5356), mouse anti-HDAC2 (catalog 5113), rabbit anti–serum response factor (anti-SRF) (catalog 5147), rabbit anti–CRE-binding protein (anti-CREB) (catalog 9197), rabbit anti–phosphorylated CREB (Ser133) (catalog 9198), rabbit anti-EGR1 (catalog 4154), and rabbit anti-GAPDH (catalog 5174) (Cell Signaling Technology); mouse anti–talin-1 (Bio-Rad, catalog MCA4770); guinea pig anti-nephrin (Progen, catalog GP-N2); rabbit anti-EGR1 (Protein Tech, catalog 55117-1-AP); Alexa Fluor 594 phalloidin (catalog A12381), Alexa Fluor 488 goat anti-mouse IgG antibody (catalog A-11001), Alexa Fluor 594 goat anti–guinea pig IgG antibody (catalog A-11076), and Alexa Fluor 594 goat anti-rabbit IgG antibody (catalog A-11037) (Invitrogen) were purchased commercially. eGFP-EGR1 plasmid was purchased from Addgene.

Techniques: Immunofluorescence, Light Microscopy, Staining, Transmission Assay