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Evidence for meiotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome heterozygous mice

Shannon Eaker, April Pyle, John Cobb and Mary Ann Handel*

Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA
* Author for correspondence (e-mail: mahandel{at}utk.edu )



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  		Fig. 1. Diagrammatic representation of a Rb chromosome meiotic pairing configuration involving chromosomes 2 (green) and 8 (yellow). The synaptonemal complex is in red, centromeres in blue. Pairing errors could give rise to aberrant alignment at MI and/or unbalanced anaphase segregation, indicated by the arrows.

 


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  		Fig. 2. Illustrations of sperm stained by the three-color FISH method. (A) A chromosomally normal sperm from a B6 mouse containing a single Y chromosome (green) and a single 8 chromosome (red). (B) An aneuploid sperm from a Rb/+ mouse containing a single Y chromosome (green) and two 8 chromosomes (red). Scale bar: 5 µm.

 


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  		Fig. 3. Air-dried meiotic metaphase I (MI) chromosome spreads labeled with chromosome paint probes (2, green; 8, red). (A) MI from a B6 mouse displaying proper pairing of homologs.

(B) Homologous pairing in an MI spermatocyte from a Rb/+ male.

(C) Failure in homologous chromosome pairing in an MI spermatocyte from a Rb/+ mouse. Scale bar: 5 µm.

 


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  		Fig. 4. Pairing abnormalities in surface-spread spermatocytes from Rb/+ mice. (A) A nucleus showing the pairing abnormalities (arrowheads) in an Rb/+ spermatocyte stained with antserum against the synaptonemal complex protein SYCP3 (red). (B) A protrusion in the pairing regions (arrowhead). Scale bars: 10 µm in A; 1 µm in B.

 


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  		Fig. 5. Confocal imaging of meiotic chromosomes and spindles from B6 and Rb/+ spermatocytes (ß-tubulin in red, phospho-histone H3 in green). (A) A MI spermatocyte from a control B6 mouse in which all chromosomes are found to be properly aligned on the metaphase plate. (B) A MI spermatocyte from a Rb/+ mouse, illustrating unaligned chromosomes (arrowheads). (C) A MI spermatocyte from a Rb/+ mouse displaying a misaligned chromosome (arrowhead) and an abnormal spindle, in which one pole is undeveloped (arrow). (D) Rb/+ MII spermatocytes depicting chromosomes lagging behind the spindle poles (arrowheads). Scale bar: 10 µm.

 


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  		Fig. 6. The frequencies of spermatogenic cell stages from B6 (white bars) and Rb/+ (black bars) adult mice. Germ cells were isolated as an enriched population from testes of three adult mice, and the number of leptotene/zygotene spermatocytes (L/Z), pachytene spermatocytes (Pach), and round spermatids (RS) were determined in a total of 250 cells per mouse. These cells represented most but not all of the cell types in the population, which also included somatic cells and elongated spermatids. Asterisks represent paired values that differ significantly (Student's t test; P=0.001 for difference in frequency of pachytene spermatocytes and P=0.008 for difference in frequency of round spermatids).

 


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  		Fig. 7. Frequencies of seminiferous tubules at indicated stages and of apoptotic tubules (red bar) in B6 (white bars) and Rb/+ (black bars) adult mice. Sectioned material was staged with Periodic Acid-Schiff reagent and Hematoxylin, and processed using the TUNEL method for detecting apoptotic cells. All stage XII tubules in Rb/+ mice were apoptotic (red). Asterisks represent paired values that differ significantly (Student's t test; P=0.035 for difference in frequency of stage VII-IX tubules and P=0.033 for difference in frequency of stage XII tubules).

 


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  		Fig. 8. Apoptotic cells in stage-XII tubule sections of B6 (A) and Rb/+ (B) 23-day-old mice. Sections were stained with Hematoxylin, Periodic Acid-Schiff reagent, and processed to observe apoptotic cells (brown cells) by the TUNEL reaction. (C) Germ cells from a preparation of micro-dissected stage XII tubule from a Rb/+ mouse. The red staining represents antibody against phosphorylated histone H3 to visualize meiotic division-stage cells and the green staining denotes apoptosis (detected by the TUNEL method). Note the unaligned chromosome (arrowhead). Scale bars: 100 µm in A,B; 10 µm in C.

 


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  		Fig. 9. CENP-E staining in prometaphase and metaphase spermatocytes from B6 and Rb/+ mice. CENP-E staining in B6 early prometaphase (A,B) and late prometaphase spermatocytes (C,D) (ß-tubulin staining in red, CENP-E staining in green). (A,C) Overlays of CENP-E and ß-tubulin staining; (B,D) CENP-E staining only. (E-G) CENP-E (green) staining in a Rb/+ MI spermatocyte, containing misaligned chromosomes (arrowheads). (E) CENP-E staining is in green and (F) DAPI staining (for DNA) is in blue; (G) overlay of E,F with MPM-2 staining in orange. Scale bars: 10 µm.

 


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  		Fig. 10. CENP-F staining in prometaphase and metaphase spermatocytes from B6 and Rb/+ mice. CENP-F staining in B6 prometaphase (A,B) and metaphase spermatocytes (C,D) (ß-tubulin staining is in red, CENP-F staining is in green). (A,C) Overlays of CENP-F and ß-tubulin staining; (B,D) CENP-F staining only. (E-G) CENP-F (green) staining in a Rb/+ MI spermatocyte, containing misaligned chromosomes (arrowheads). (E) CENP-F staining is in green and (F) DAPI staining (for DNA) is in blue; (G) overlay of E,F with MPM-2 staining in orange. Scale bars: 10 µm.

 

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