Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 2. Loss of SGO2 at the pericentromeric bridge with increased inter-sister kinetochore distance (A) Schematic summarizing SGO2 localization in metaphase I oocytes from younger women. (B) Inter-sister kinetochore distance (white dashed arrows) was determined on metaphase I chromosomes and related to the presence of the SGO2 bridge.

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques:

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 3. Loss of cohesion in metaphase II oocytes with age and absence of bridge SGO2 (A) Representative image showing presence of single chromatids (arrows) in human metaphase II oocytes from an older (age 40) woman, compared with a younger (age 30) woman without single chromatids. White boxes with dashed lines indicate examples of paired and single chromosomes in oocyte 2. SGO2 (green), inner kinetochores (CENP-C; magenta), microtubules (a-tubulin; orange), and chromosomes (Hoechst; blue) are shown. (B) Increase of single chromatids in metaphase II oocytes with maternal age. The number of single chromatids was scored relative to woman’s age. Data were fit to a sigmoidal, 4 parameter logistic curve (R2 = 0.57). Oocytes used in representative images are labeled in the graphs. (C) Increased number of single chromatids in metaphase II oocytes with an increased fraction of sister chromatid pairs lacking a SGO2 bridge. Data were fit to a sigmoidal, 4 parameter logistic curve (R2 = 0.60). (D) The relative intensity of the centromeric pool of SGO2 between paired and single chromatids was measured only from oocytes that had single chromatids. SGO2 intensity was measured in arbitrary units (a.u.) relative to the kinetochore markers CENP-C (p = 0.96, Mann-Whitney test) and CREST (p = 0.24, Mann- Whitney test). Plots show median (dashed black line) and 25th and 75th percentiles (dotted black lines). p values were calculated using the Mann-Whitney test. n.s., not significant. See also Figure S3.

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques: Labeling, MANN-WHITNEY

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 4. Co-localization of PP2A and cohesin with the SGO2 pericentromere bridge on human metaphase II chromosomes (A and B) Chromosome spreads of metaphase II-arrested human oocytes were stained with antibodies against SGO2 (green), inner kinetochores (CENP-C, magenta), and cohesin (REC8, orange). Chromosomes were stained with Hoechst (blue). (A) Representative images with white dashed line boxes indicating representative chromosome figures shown at higher magnification below. (B) Localization of REC8 at the pericentromere bridge was scored for sister chromatid pairs with and without a SGO2 bridge. Schematic representations of the data are shown below. Statistical analyses were performed using the chi-squared test (****p < 0.0001). (C and D) Chromosome spreads of metaphase II-arrested human oocytes were stained with antibodies against SGO2 (green), inner kinetochores (CENP-C, magenta), and PP2A (orange). Chromosomes were stained with Hoechst (blue).

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques: Staining

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 6. Dependence of SGO2 localization on BUB1 activity (A) Scheme of the experiment. Metaphase II oocytes from women aged % 36 years were treated with 10 mM BAY-320 (to inhibit BUB147) or DMSO (control) overnight and fixed. (B) Representative images of control and Bay-320-treated metaphase II oocytes after immunostaining with antibodies against SGO2 (green), CENP-C (inner kinetochores, magenta), and a-tubulin (microtubules, orange). White boxes with dashed lines indicate chromosomes that have been further magnified below. (C) The percentage of chromatids per oocyte with SGO2 localization at the pericentromeric bridge was scored in control and BAY-320-treated metaphase II oocytes (****p < 0.0001, Mann-Whitney test). (D) The relative intensity of the centromeric pool of SGO2 in metaphase II oocytes from control and BAY-320-treated oocytes were measured relative to CENP-C. (****p < 0.0001, Mann-Whitney test.) Plots show median (dashed black line) and 25th and 75th percentiles (dotted black lines). (E) Schematic summarizing a model for MPS1 and BUB1 in SGO2 localization in metaphase II oocytes from younger women. See also Figure S6.

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques: Activity Assay, Control, Immunostaining, MANN-WHITNEY