Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: (a) top panel: Chemical structures of N-cadherin antagonists CRS-066; LCRF-0006 and analogues RB-192 and RB-028. Active side-chain or modified side-chain depicted by highlighted box. middle panel: Average cell adhesion index values of pre-ovulatory COCs (11h post-hCG) interacting with a fibronectin substrate in presence of vehicle or N-Cadherin antagonists CRS-066, LCRF-0006, and sidechain modified analogues RB-192 and RB-028 or vehicle at respective doses. Cell indices were determined using the xCELLigence Impedance system over 15h (n=3 independent experiments with 2 technical replicates per treatment). bottom panel: Mean ± SD adhesion index values at 6 h and IC 50 values of respective drugs calculated from dose-response curve and compared using one-way ANOVA. ( b and d) Representative confocal images of N-cadherin adherens junctions on SK-OV-3 cells treated with N-cadherin antagonists CRS-066 (0.1-1.0 μM), LCRF-0006 (36-360 μM) or vehicle for 24h. (N=3). Scale bar: 40μM.( c and e ) Quantification of N-cadherin adherens junctions. Mean of total pixel intensity in red channel, +/- SEM of at least 50 cells. N=3 independent experiments. Statistical analyses with two-tailed unpaired t-test. * denotes <0.01.( f ) Bright-field images of spheroid formation in 67NR mouse mammary cell line expressing ectopic Cdh2 were treated with CRS-066 or vehicle at respective time-points. Cells were seeded at 2000 cells per well, and formation of spheroids was assessed by imaging every hour for 6h. ( g ) Mean +/- SEM of spheroid area in 67NR-Cdh2 cells treated with either vehicle or increasing doses of CRS-066 (0-2 μM) over 6h.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Analogues, Modification, Two Tailed Test, Expressing, Imaging

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: ( a - c ) Time-course of Cdh2, ctnnb1 and Cdh1 mRNA expression in isolated granulosa cell (GCs) or Cumulus oocyte complexes (COCs) from mouse ovaries at indicated time-points after eCG and hCG stimulation of folliculogenesis and ovulation (N=3 animals per time point). Cdh2 and Ctnnb1 levels are high in GC and COC throughout folliculogenesis, with a transient drop in Cdh2 level 12 h after ovulation stimulus, while E-Cadherin was high in COCs and significantly reduced by ovulation stimulus. The levels shown of the indicated mRNAs were determined by TaqMan qPCR normalised to Rpl19. ( d ) Immunofluorescent staining of N-Cadherin, βcatenin and E-cadherin throughout ovarian folliculogenesis. Confocal images of mouse ovarian sections obtained from eCG primed mice and stained using anti N-Cadherin (left panel), anti-β-catenin (middle panel) and E-cadherin (right panel). DNA is counterstained with Hoechst. Arrows indicate presence of N-Cadherin and β-catenin at granulosa-granulosa cell junctions in secondary and antral follicle stages. Arrowheads indicate presence of N-cadherin and β-catenin at oocyte-cumulus interface. High magnification images show transzonal projections extending from cumulus cells and anchored to oocyte membrane Scale bar: 50 µM. ( e ) Whole-mount immunofluorescent staining showing co-localization of N-cadherin (green) and E-cadherin (red) at the oocyte plasma membrane in mouse COC from antral follicles of eCG primed mice. N-cadherin is also evident on cumulus cell surfaces and transzonal projections. Cumulus cell and oocyte nuclear DNA is counterstained with Hoechst. Scale bar: 50 µM. ( f ) Wholemount immunostaining shows loss of β-catenin and E-cadherin at the oocyte plasma membrane after treatment with CRS-066. COCs obtained from antral follicles of eCG primed mice and treated with CRS-066 or vehicle for 4h. COCs were fixed and stained with anti-E-cadherin (green) and anti-β-catenin (red). DNA was counterstained with Hoechst. Scale bar: 50 µM.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Isolation, Staining, Membrane, Clinical Proteomics, Immunostaining

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: COCs from eCG primed mice were treated with LCRF-0006 (36-360 uM) or CRS-066 (0,1-1 uM) during in vitro maturation (EGF and FSH stimulated) and cumulus expansion was assessed after 12 h or gene expression assessed after 10 h IVM. ( a and d ) Representative bright-field images of COCs after 12 h IVM treated with LCRF-0006 or CRS-066. Scale bar: 10µm. ( b and e ) Mean ±SEM of Cumulus expansion indices from a and d n= >20 COCs per experiment. N=4 independent experiments, * = P<0.05, ** = P<0.01. ( c and f ) Effect of N-cadherin antagonist treatment during IVM (10 h) on the expression of key genes involved in COC expansion during IVM. Mean± SEM. N=3 independent experiments. Statistical testing with one-way ANOVA * P<0.05, **P<0.01. ( g and h ) Gene ontology enrichment of biological pathways and molecular functions of significantly differentially down-regulated genes identified in RNA-Seq analysis of COCs after CRS-066 (0.3 μM) treatment compared to vehicle treatment.. All data are presented as the ratio of CRS-066 over vehicle (N=3). ( I and j ) Gene set enrichment analysis plot (GSEA) demonstrating the upregulation of Ctnnb1 and Hippo signalling pathways in CRS-066 treated COCs versus vehicle treated COCs. Net enrichment score (NES) values are shown. N=3 independent biological replicates. ( k ) Heat map representing the relative expression profiles of transcripts involved in Wnt\β- catenin, Hippo\YAP and ovarian signalling axes.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: In Vitro, Gene Expression, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: ( a ) Ovulation rate of 21d old mice treated with CRS-066 (50mg/kg) or vehicle (7.5% DMSO in 0.9% saline). COCs in oviducts counted 16h after hCG injection. Graph represents mean ±SEM from N=6 animals; ***p<0001 (unpaired two-tailed t-test). ( b ) Histology of ovaries by Haematoxylin & Eosin staining. CL indicate corpus leuteum; arrows indicate trapped oocytes in CL. Scale bar: 100µm. ( c ) Hierarchical clustering of RNA-sequencing analysis results shows differentially expressed genes between CRS-066 and vehicle treated mice (N=6 mice per treatment). ( d and e ) Gene ontology enrichment of biological pathways (D) and molecular functions (E) of significantly differently down-regulated genes in CRS-066 treated mouse ovaries compared to vehicle treated ovaries. ( f and g ) Gene set enrichment analyses plot (GSEA) shows downregulation of Ctnnb1 and Hippo signalling pathways in CRS-066 treated mice ovaries compared to vehicle treated ovaries. Net enrichment score (NES) values are shown. ( h ) Heatmap representing the relative expression profiles of transcripts involved in Wnt\β-catenin, Hippo\YAP and ovarian signalling axes in CRS-066 treated ovaries compared to vehicle. N=3 biological replicates. ( i - k ) Relative mRNA expression of key genes involved in gonadotrophin signalling and oocyte function (i), COC expansion and ovulation (j) or folliculogenesis (k) h in ovaries treated with CRS-066 compared to vehicle treatment and determined by qRT-PCR. Bar graph show mean+/- SEM. N=6 ovaries from independent CRS or vehicle treated mice. Statistical testing with Student’s t-test; *P < 0.05; ** P<0.01; **** P <0.00001. ( l ) Follicle counts at primary, secondary, pre-antral, antral and ovulatory stages in ovaries from mice treated with either CRS-006 (50mg/kg) or vehicle control (7.5% DMSO). N=3 mice/treatment/time-point. ( m ) Representative follicle morphology H&E (left) section and N-cadherin immunofluorescence (right) section in mice treated with either CRS-066 or vehicle. H&E and immunoflourescence highlight disorganised granulosa cells organisation. Asterisks indicate loss of transzonal projections between oocyte and cumulus cells. Scale bar: 30µm. ( n ) Representative confocal immunofluorescent images of mouse ovaries stained with anti-cleaved caspase 3 and anti-Ki-67. Scale bar: 30µm. ( o ) Relative mRNA expression of key genes involved in oocyte growth and ovulation in CRS-066 or vehicle treated mice (N=3/ treatment) at either 44h post eCG or 11h post hCG.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Saline, Injection, Two Tailed Test, Staining, RNA Sequencing, Expressing, Quantitative RT-PCR, Control, Immunofluorescence

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: (a) qPCR analysis of relative mRNA expression of Cdh2, Areg, Ptgs2 and Cyp19a1 in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), n=6 individual animals, *p<0.05. ( b-e ) Immunofluorescent analysis of N-cadherin protein in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), showing mosaic depletion of N-cadherin in granulosa cells of mutant follicles. Arrows indicate mosaic regions with persistent N-cadherin. ( f ) Ovulation rate of 21d old mice with indicated control or granulosa-specific mutant genotypes. COCs in oviducts counted 16h after hCG injection. Graph represents mean ±SEM from N=4 and 7 animals respectively; **p<0.01 (unpaired two-tailed t-test). ( g ) Histology of ovaries by Haematoxylin & Eosin staining. Scale bar: 100µm.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Control, Mutagenesis, Injection, Two Tailed Test, Staining