Figure 4.

Bivalent Cohesion Is Maintained without Detectable Turnover of Rec8-Cohesive Structures for Months during the Dictyate Arrest

(A) Rec8-Myc is transcribed in adult ovary. Detection of mRNA for Nobox, Smc1β, Rec8, and Rec8-Myc by RT-PCR from adult ovary. RT, reverse transcriptase. See also Figure S4.

(B) Rec8-Myc is synthesized in oocytes from adult Rec8^TEV/TEV(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P female analyzed 2 months post-4-OHT. Metaphase I chromosome spread showing expression and localization of Rec8-Myc to bivalent chromosomes. Left panel shows 3D surface plots of Rec8-Myc pixel fluorescence intensities. Centromeres are marked by ACA. Oocytes analyzed from top to bottom: n = 11, 7, 16. Inset has been brightness and contrast enhanced equally in all three panels. Scale bar, 20 μm; inset scale bar, 5 μm.

(C) Timing of cohesion rescue assay utilizing Dppa3-MCM-P to activate Rec8-Myc in adult female mice. Green, meiotic DNA replication; beige, homologous recombination; pink, dictyate stage.

(D and E) Representative still images of oocytes isolated from Rec8^TEV/TEV(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P females microinjected with mRNA encoding H2B-mCherry, CenpB-EGFP, and TEV protease.

(D) Bivalents are retained in oocytes microinjected with mutant TEV mRNA.

(E) TEV protease converts bivalents to chromatids in cells with successful Stop cassette deletion. Single-cell genotyping was performed after live-cell imaging. Scale bar, 20 μm.

(F) Quantification of cohesion rescue assay in oocytes from Rec8^TEV/TEV(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P females with successful Stop cassette deletion used 2 or 4 months post-4-OHT treatment. Oocytes were obtained from >3 females; n = number of oocytes analyzed.

See also Figure S3; Table S1; Movies S1 and S2.