N-cadherin controls adhesive capacity of ovulating COCs.

(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 IC50 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.

N-cadherin maintains oocyte-cumulus cell interaction.

(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.

N-cadherin antagonists block cumulus expansion in mouse COCs.

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.

N-cadherin antagonists block meiotic maturation in mouse COCs.

COCs from eCG primed mice were treated with LCRF-0006 (36-360 uM) or CRS-066 (0,1-1 uM) during in vitro maturation (12 h EGF and FSH stimulated), then oocytes were denuded and meiotic stage was assessed by labelling actin (phalloidin, red), spindles (tubulin IF, green) and DNA (Hoechst, blue). n= >20 COCs per experiment; N=4 independent experiments. (a and d) Representative bright field images of denuded mouse COCs showing oocyte and polar body morphology after vehicle, CRS-066 or LCRF-0006 treatment. Scale bar: 5µM. (b and e) Representative confocal fluorescent images of denuded mouse oocytes after IVM treated with LCRF-0006 or CRS-066 as indicated, showing polar bodies, spindle structure or germinal vesicle morphology typical in each treatment condition. Scale bar: 20µM. Pb indicates polar body formation; Oo indicates Oocyte. (c and f) Percent of oocytes at each stage of meiotic progression from n>20 oocytes per experiment in 4 independent experiments. MI defined by the any evidence of tubulin staining in the first metaphase spindle with no polar body extrusion; MII defined by presence of a polar body and MII metaphase plate. GV defined by oocyte DNA lacking any tubulin staining to indicate MI spindle formation.

N-Cadherin antagonist CRS-066 blocks ovulation in vivo.

(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.

Granulosa conditional Cdh2 null mutation disrupts ovarian gene expression and blocks ovulation.

(a) qPCR analysis of relative mRNA expression of Cdh2, Areg, Ptgs2 and Cyp19a1 in ovaries of control (Cdh2Fl/+ ; Amhr2Cre) and granulosa-specific Cdh2 null mutants (Cdh2Fl/Fl ; Amhr2Cre), n=6 individual animals, *p<0.05. (b-e) Immunofluorescent analysis of N-cadherin protein in ovaries of control (Cdh2Fl/+ ; Amhr2Cre) and granulosa-specific Cdh2 null mutants (Cdh2Fl/Fl ; Amhr2Cre), 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.