Bmi1 Deficient Mice Exhibit Male Infertility

Abstract

Previous studies have demonstrated that the polycomb repressor Bmi1 is universally expressed in all types of testicular cells and might regulate the spermatogonia proliferation, however, it is unclear whether Bmi1 plays a critical role in maintaining normal male fertility in vivo. To answer this question, we first confirmed that Bmi1 is universally expressed in all types of testicular cells and found that the gene relative expression levels of Bmi1 in testis were the highest relative to other organs. Then we investigated the role of Bmi1 in maintaining normal male fertility using Bmi1 knockout male mouse model. Our results demonstrated that Bmi1 deficiency resulted in totally male infertility with smaller testis, severe oligospermia and sperm malformation. Mechanistically, decreased serum testosterone levels with down-regulating 3βHSD and 17βHSD expression levels, reduced germ cell proliferation, increased germ cell apoptosis with up-regulating p16, p19, p53 and p21 expression levels, increased reactive oxygen species (ROS) and H₂O₂ levels with down-regulating gene expression levels of anti-oxidant enzymes, and increased 8-OHdG and γ.H2AX positive cells in testis were observed in Bmi1 deficient mice compared with wild-type mice. These results indicate that Bmi1 deficiency results in male infertility by reducing testosterone syntheses, increasing oxidative stress and DNA damage, activating p16 and p19 signaling pathway, inhibiting germ cell proliferation and inducing germ cell apoptosis and sperm malformation. Thus, Bmi1 may be a novel and potential target for the clinic treatment of male infertility.

Fig 1

Bmi1 expression and localization in testes. (A) Real-time RT-PCR analysis of multiple organs extracts from 7-week old male mice for Bmi1 expression. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to liver. Representative micrograph of testicular sections from 7-week old WT mice for (B) Bmi1 and PLZF double immunofluoresent staining and (C) Bmi1 immunohistochemical staining. Magnification is ×400 in B and C. Stars: spermatogonia. Arrows: sertoli cells.

Fig 2

Bmi1 deficiency induces male infertility with severe oligospermia in mice. (A) Litter size. (B) CASA analysis of sperm counts in caudal of epididymis from 7-week old male mice. (C) Testis size and (D) weight from 7-week-old mice. Representative micrographs of H&E staining for (E) testicular sections, (F) epididymis sections from 7-week-old mice. Magnification is ×400 in E and F. Each value is the mean ± SEM of determinations in 13-15 mice of each group. * * *: P < 0.001 compared with WT mice.

Fig 3

Effect of Bmi1 deficiency on leydig cells and testosterone synthesis. (A) Serum testosterone levels from 7-week-old mice. (B) Representative graphs of flow cytometry analysis of testicular germ cells stained for 3βHSD. (C) Quantitative percentage of 3βHSD positive cells relative to total germ cells from 7-week-old mice. (D) Western blot and (F) Real-time RT-PCR analysis of testis extract from 7-week-old mice for expressions of Hsd3b1 and Hsd17b1. Messenger RNA expression determined by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to WT mice. (E) 3βHSD and 17βHSD protein levels were assessed by densitometry analysis calculated as a ratio relative to β-actin protein levels and expressed relative to levels of WT mice. Each value is the mean ± SEM of determinations in 5 mice of each group. *: P < 0.05compared with WT mice.

Fig 4

Effect of Bmi1 deficiency on germ cell proliferation, apoptosis and sperm morphology. Representative micrographs of testicular sections from 7-week-old mice stained (A) immunohistochemically for Ki67, (C) for TUNEL. The numbers of (B) Ki67 and (D) TUNEL positive cells per tube. (E) Representative micrograph of sperms from 7-week-old Bmi1^-/- mice stained for H&E (a: normal head b: irregular head, c & d: big head) (F) Evaluated abnormality rate of sperm from 7-week-old mice. (G) Representative micrographs of ultrastructure of sperms photoed by Electron microscopy. (H) Real-time RT-PCR analysis of testis extracts from 7-week-old mice for Tnp1, Tnp2, Prm1, Prm2, H1fnt and Spem1 expressions. Messenger RNA expression determined by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to WT mice. (I) Representative immunofluorescent micrographs of sperms stained with Mito-tracker. Magnification is ×400 in A, C, E and I, and is ×15000 in G. Each value is the mean ± SEM of determinations in 5 mice of each group. *: P < 0.05; **: P < 0.01; * * *: P < 0.001 compared with WT mice.

Fig 5

Effect of Bmi1 deficiency on p16 /p19 signaling pathway in testes. (A) Representative micrographs of testicular sections from 7-week-old mice stained immunohistochemically for (A) p16 and (C) p-p53. Magnification is ×400 in A and C. Quantitative numbers of (B) p16 and (D) p-p53 positive cells per tube. (E) Western blot analysis of testis extracts from 7-week-old mice for expressions of p19, p53, p21 and p16. Each value is the mean ± SEM of determinations in 5 mice of each group. *: P < 0.05compared with WT mice.

Fig 6

Effect of Bmi1 deficiency on oxidative stress status and DNA damage in testes. (A) Representative graphs of flow cytometry analysis of testicular germ cells from 7-week-old mice stained for DCFDA. (B) ROS levels were assessed by mean fluorescence intensity analysis and presented relative to levels of WT mice. Biochemistry analysis of testis extracts from 7-week-old mice for (C) H₂O₂ levels and (D) total antioxidant capacity. (E) Real-time RT-PCR analysis of testis extracts from 7-week-old mice for expressions of Gpx1, Gpx4, Gsr, Cat and Txnrd1. Messenger RNA expression determined by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to WT mice. (F) Representative micrographs of testicular sections from 7-week-old mice stained immunohistochemically for (F) 8-OHdG and (H) γ.H2AX. Magnification is ×400 in F and H. Quantitative numbers of (G) p16 and (I) γ.H2AX positive cells per tube. Each value is the mean ± SEM of determinations in 5 mice of each group. *: P < 0.05; * * *: P < 0.001 compared with WT mice.