Paternal USP26 Mutations Raise Klinefelter Syndrome Risk in the Offspring of Mice and Humans (2021)
Figure 5

Figure 5 - Usp26-deficient mice produce XY aneuploid spermatozoa.

A, B Laggard chromosomes were observed in the metaphase I of 6-month-old Usp26-/Y spermatocytes. Immunofluorescence analysis of tubulin (green) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the laggard chromosomes.

(C) The proportion of metaphase I spermatocytes exhibiting laggard chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice spermatocytes (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Parafﬁn sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.

E The protein level of three known SAC proteins was reduced in 2-month-old and 6-month-old Usp26−/Y mouse testes. Immunoblotting of MAD2, BUBR1, PLK1 and USP26 was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26-/Y testes. H3Ser10p and Tub served as loading control.

F Usp26-deficient mice produced XY aneuploid round spermatids. FISH analysis of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y round spermatids. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.

G Quantification of different types of round spermatids in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y (n= 3 independent experiments), Usp26-/Y mice (n= 4 independent experiments). P=0.0026 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. P=0.0216 for O spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice.

H Usp26-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.

I Quantification of different types of spermatozoa in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n= 5 independent experiments). P=0.0195 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. Data are presented as means ± SD.

J Usp26-deficient mice could not produce Chr 8, Chr 10 aneuploid spermatozoa. FISH assay of Chr 8 (green) and Chr 10 (red) was performed in 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue).

K Quantification of different types of spermatozoa in 6-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments). Data are presented as means ± SD.

L The disruption of Usp26 produces XY aneuploid spermatozoa and results in the production of 41, XXY offspring.

Data information: In (C), (G), (I) and (K), data are presented as mean ± SD. **P < 0.01 (Student's t-test). Numerical source data for panels C/G/I/K can be found in Table EV5.