105522)  (Addgene inc)

Journal: bioRxiv

Article Title: Hybrid female sterility due to cohesin protection errors in oocytes

doi: 10.1101/2025.02.16.638358

Figure Lengend Snippet: ( A ) Phylogenetic tree of mouse species (myr, million years) and schematic of the hybrid mouse system to study hybrid incompatibility in female meiosis. ( B ) 6-month fertility test from indicated genotypes. Age-matched domesticus (dom), spicilegus (spi) and the hybrid female (F1 hybrid) were used. Each column represents the number of pups per litter for each breeding cage (n = 4 breeding cages per genotype). Note that two F1 female hybrids gave birth to one pup each, which died within few days after the birth. ( C ) Schematic of meiotic chromosome segregation. Cohesin is initially loaded along the chromosome axes and holds sister chromatids together. Cohesin cleavage along the chromosome arm at anaphase I allows the segregation of homologous chromosomes. During metaphase II, sister chromatids maintain their cohesion by the residual cohesin at the pericentromere. Cleavage of this remaining cohesin at anaphase II leads to sister chromatid segregation. ( D ) DNA was visualized in domesticus and hybrid oocytes by incubating with SPY-DNA or expressing the Separase biosensor, H2B-mScarlet-Rad21- mNeonGreen (see ) to live-image anaphase I; the mScarlet images are shown in the figure; PB, polar body; dashed lines, oocyte cortex. ( E ) Anaphase chromosome lagging rates in D were quantified (n = 68 and 65 oocytes for domesticus and hybrid, respectively); red lines, mean; unpaired two-tailed t test was used for statistical analysis; ** P <0.01. ( F ) Chromosome spreads were performed at metaphase II using domesticus , spicilegus and hybrid oocytes and stained for HEC1 and REC8 (right); orange arrowhead, univalents; white arrowhead, bivalents. ( G ) The number of bivalents per egg and the percentage of eggs with cohesin along the chromosome axes were quantified using the images in F (left bottom, n = 13, 13, and 13 eggs for domesticus , spicilegus , and hybrid); each dot in the graph represents a single egg; red line, median. ( H ) domesticus and hybrid oocytes expressing mCherry-Trim21 with or without the anti-REC8 antibody were fixed at metaphase II and stained for ACA (centromere). The percentage of meiosis II eggs with >1 bivalent, >1 precocious separated sister chromatids (PSSC), and normal univalents were quantified (n = 12, 10, 16, and 18 eggs for domesticus + TRIM21, domesticus + TRIM21 + anti-REC8, hybrid + TRIM21, and hybrid + TRIM21+anti- REC8); scale bars, 5 µ m. Schematics in A and C were created using BioRender.

Article Snippet: GV-intact prophase I oocytes were microinjected with ∼5 pl of cRNA or antibodies in M2 containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). cRNA used for microinjections were Egfp-Bub1 ( M. m. domesticus or P. maniculatus bairdii BUB1 fused with EGFP at the N-terminus, 1184 and 950 ng/µl, respectively), mCherry-Trim21 (Addgene cat# 105522, M. musculus domesticus TRIM21 fused with mCherry at the C-terminus, 1500 or 3000 ng/µl for BUB1 and REC8 TrimAway, respectively), and hH2B-mScarlet-hRad21-mNeonGreen (Separase sensor, pNM853, human H2B fused with human Rad21(142-476 a.a.) at the C-terminus with the Rad21 fragment flanked by two fluorescent proteins, mScarlet and mNeonGreen, 750 ng/µl).

Techniques: Expressing, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Hybrid female sterility due to cohesin protection errors in oocytes

doi: 10.1101/2025.02.16.638358

Figure Lengend Snippet: ( A ) Chromosome spreads were performed at metaphase I using domesticus , spicilegus , and hybrid oocytes and stained for BUB1. Line scans were performed to quantify BUB1 signal intensities along the chromosome arm starting from the centromere. Line graph shows the mean values of BUB1 intensity along the chromosome arm with the shades representing standard deviation (n = 26, 36, and 60 chromosomes for domesticus , spicilegus , and hybrid). The enlarged area of the graph highlights the difference of BUB1 intensity along the chromosome arm. ( B ) Chromosome spreads were performed at metaphase I using domesticus , spicilegus , and hybrid oocytes and stained for H2ApT121. Graph is the quantification of chromosomal H2ApT121 signal intensities per oocyte. (n = 13, 6, and 17 oocytes for domesticus , spicilegus , and hybrid); each dot in the graph represents a single oocyte; red line, median. ( C ) Ovarian granulosa cells from domesticus , spicilegus , and hybrid were fixed and stained for BUB1 and H2ApT121. Line graph is the quantification of BUB1 and H2ApT121 signal intensities along the chromosome arm starting from the centromere. Lines indicate the mean values of BUB1 and H2ApT121 intensities, and the shades represents standard deviation (n = 64, 93, and 102 chromosomes for domesticus , spicilegus , and hybrid). ( D ) domesticus and hybrid oocytes expressing mCherry-TRIM21 with the control IgG or the anti-BUB1 antibodies were fixed at metaphase II and stained for ACA. The percentage of eggs with >1 bivalent, >1 precocious separated sister chromatids (PSSC), and normal univalents were quantified (n = 21, 26, 38, and 54 eggs for domesticus + TRIM21 + IgG, domesticus + TRIM21 + anti-BUB1, hybrid + TRIM21 + IgG, and hybrid + TRIM21 + anti- BUB1). ( E ) domesticus oocytes expressing EGFP-BUB1 derived from domesticus or Peromyscus maniculatus were fixed at metaphase II and stained for EGFP. The percentage of meiosis II eggs with >1 bivalent was quantified (n = 31, 26, 39 oocytes for control, domesticus EGFP-BUB1, Peromyscus EGFP-BUB1, respectively); each dot in the graph represents a single oocyte; red line, mean; unpaired two-tailed t test was used for statistical analysis; ** P <0.01, ** *P <0.001; scale bars, 5 µ m.

Article Snippet: GV-intact prophase I oocytes were microinjected with ∼5 pl of cRNA or antibodies in M2 containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). cRNA used for microinjections were Egfp-Bub1 ( M. m. domesticus or P. maniculatus bairdii BUB1 fused with EGFP at the N-terminus, 1184 and 950 ng/µl, respectively), mCherry-Trim21 (Addgene cat# 105522, M. musculus domesticus TRIM21 fused with mCherry at the C-terminus, 1500 or 3000 ng/µl for BUB1 and REC8 TrimAway, respectively), and hH2B-mScarlet-hRad21-mNeonGreen (Separase sensor, pNM853, human H2B fused with human Rad21(142-476 a.a.) at the C-terminus with the Rad21 fragment flanked by two fluorescent proteins, mScarlet and mNeonGreen, 750 ng/µl).

Techniques: Staining, Standard Deviation, Expressing, Control, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Hybrid female sterility due to cohesin protection errors in oocytes

doi: 10.1101/2025.02.16.638358

Figure Lengend Snippet: ( A ) Phylogenetic tree of the Mus , Rattus , and Peromyscus genera (myr, million years) and schematic of the Peromyscus hybrid mouse system. ( B ) P. maniculatus , P. polionotus , and their hybrid oocytes (with or without the EGFP-BUB1 expression) were matured to meiosis II, fixed and stained for HEC1. The numbers in the DNA images indicate the number of split sister chromatids in the egg. ( C ) Graph shows the quantification of the number of separated sister chromatids in each meiosis II egg (n = 162, 171, 147, and 50 eggs for P. maniculatus , P. polionotus , hybrid, and hybrid + EGFP-BUB1, respectively); each dot represents an independent experiment; bars and error bars represent mean and standard deviation, respectively. ( D ) P. maniculatus , P. polionotus , and hybrid metaphase II eggs were fixed and stained for REC8 and HEC1. ( E ) Bar graph shows the proportion of meiosis II eggs with centromeric REC8 signals in each genotype (n = 32, 21, and 40 eggs for P. maniculatus , P. polionotus , and the hybrid); each dot represents an independent experiment; bars and error bars represent mean and standard deviation, respectively; unpaired two-tailed t test was used for statistical analysis. Images from were used for the quantification. Dot plot is a quantification of sister-kinetochore distance at metaphase II; images from were used for the quantification; red line, median; Mann-Whitney test was used for statistical analysis. ( F-H ) P. maniculatus , P. polionotus , and hybrid oocytes were fixed at metaphase I and stained for ACA together with PP2A (f), H2ApT121 (g), or BUB1 (h). The graphs show the quantification of centromeric signal intensities for PP2A (f, n = 678, 403, and 448 centromeres for P. maniculatus , P. polionotus , and the hybrid), H2ApT121 (g, n = 473, 301, and 322 centromeres for P. maniculatus , P. polionotus , and the hybrid), and BUB1 (h, n = 805, 394, and 1213 centromeres for P. maniculatus , P. polionotus , and the hybrid); each dot represents a single centromere; red line, median; Mann-Whitney test was used for statistical analysis. **P <0.01, ****P <0.0001; scale bars, 5 µ m.

Article Snippet: GV-intact prophase I oocytes were microinjected with ∼5 pl of cRNA or antibodies in M2 containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). cRNA used for microinjections were Egfp-Bub1 ( M. m. domesticus or P. maniculatus bairdii BUB1 fused with EGFP at the N-terminus, 1184 and 950 ng/µl, respectively), mCherry-Trim21 (Addgene cat# 105522, M. musculus domesticus TRIM21 fused with mCherry at the C-terminus, 1500 or 3000 ng/µl for BUB1 and REC8 TrimAway, respectively), and hH2B-mScarlet-hRad21-mNeonGreen (Separase sensor, pNM853, human H2B fused with human Rad21(142-476 a.a.) at the C-terminus with the Rad21 fragment flanked by two fluorescent proteins, mScarlet and mNeonGreen, 750 ng/µl).

Techniques: Expressing, Staining, Standard Deviation, Two Tailed Test, MANN-WHITNEY