Fig 1: Localization of LRRCC1-Gfp variants does not differ from WT.(a) Schematic of LRRCC1 protein with conserved domains and variants labeled. h is human protein sequence and m is mouse protein sequence. Image created by BioRender. (b) Confocal images of LRRCC1 variant localization during meiotic maturation: GFP-LRRCC1 (WT), GFP-LRRCC1H69Q (H69Q), GFP-LRRCC1L780P (L780P), and GFP-LRRCC1R802Q (R802Q). In overlay, LRRCC1 (GFP; gray), DNA (DAPI; blue), spindle (α-tubulin; green) and aMTOC (PCNT; magenta). Red arrows indicate LRRCC1 localization. n, total number oocytes per meiotic stage examined in 2 experimental replicates. Scale bar: 10μm. (c) Western blot detecting GFP in uninjected control oocytes and oocytes injected with indicated constructs. α-Tubulin served as loading control. 30 oocytes per group. (d) Quantification of relative GFP/α-tubulin ratio. Bars and error bars indicate ± SEM. One-way ANOVA used to determine significance. ns, not significant. Full length blot is Figure S1.
Fig 2: LRRCC1H69Q variant causes increased egg aneuploidy.(a) Schematic of experimental design to assess oocyte maturation and aneuploidy. Image created by BioRender. (b) Confocal images from in situ chromosome counting assay on metaphase II eggs expressing the indicated LRRCC1 constructs. Eggs were stained to detect centromeres (anti-centromeric antigen, ACA; red) and DNA (DAPI; gray). Extra chromosome indicated in yellow. n, total number oocytes examined in 3 experimental replicates. Scale bar: 5μm. (c-d) Frequency of polar body extrusion and aneuploidy in oocytes expressing GFP control and LRRCC1 WT and variants as indicated. One-way ANOVA used to determine significance. Bars and error bars indicate ± SEM. ns, not significant; **P<0.01.
Fig 3: LRRCC1H69Q variant causes smaller MI spindles and misaligned chromosomes.(a) Schematic of experimental design to assess MI spindle phenotypes. Image created by BioRender. (b) Confocal images of MI oocytes expressing the indicated LRRCC1 constructs stained to detect the spindle (α-tubulin; green), DNA (DAPI; blue) and LRRCC1 (GFP; gray). n, total number oocytes examined in 3 experimenal replicates. Scale bar: 10 μm. (c-d) Quantification of the relative MI spindle length and spindle volume. (e) Quantification of the relative MI metaphase plate width signifying chromosome alignment. Mean indicated ± SD. One-way ANOVA used to determine significance. ns, not significant; *P < 0.05, ***P < 0.001.
Fig 4: Expression of exogenous LRRCC1 protein.a) Schematic of workflow and oocyte meiotic maturation. Image created by BioRender. b) Confocal images of LRRCC1-Gfp expression in mouse oocytes (GFP; gray), DNA (DAPI; blue), spindle (α-tubulin; green) and aMTOCs (pericentrin, PCNT; magenta). Red arrows indicate LRRCC1 localization. Yellow arrows indicate LRRCC1 localization in polar body. n, total number oocytes examined for each meiotic stage in 2 experimental replicates. Scale bar: 10μm.
Fig 5: LRRCC1H69Q variant alters aMTOC clustering.(a) Experimental design to assess MI spindle aMTOC phenotype. Image created by BioRender. (b) Confocal images of MI oocytes expressing the indicated LRRCC1 constructs stained to detect the aMTOCs (PCNT; magenta), spindle (α-tubulin; green) and DNA (DAPI; blue). n, total number oocytes examined in 3 experimental replicates. Scale bar: 10 μm. (c-e) Quantification of the relative metaphase I spindle aMTOC pixel intensity, relative foci volume and relative foci count. Mean indicated ± SD. Student’s t-test used to determine significance. ns, not significant. *P < 0.05.
Supplier Page from Sino Biological, Inc. for Human LRRCC1 Gene ORF cDNA clone in cloning vector