Histone hyperacetylation disrupts spermatogonial stem cells homeostasis and impairs spermiogenesis

Abstract

Background: It is well-known that epigenetic regulation is involved in the negative effects of environmental physical, chemical and biological factors exposures on organs. To investigate the effects and mechanisms of histone hyperacetylation caused by environmental stress on spermatogenesis, we used the histone deacetylase inhibitor Panobinostat (PANO) to establish the hyperacetylation models in mice.

Methods: To investigate the effects of PANO on testicular function of male reproductive system, we conducted the evaluation of sperm quality parameters in mice. The morphological changes of testes were observed through hematoxylin and eosin (H&E) staining on paraffin sections and quantified by stereological measurements after treatment to explore the cause of infertility and figure out if PANO has any effect on the reproductive system of male mice. Immunofluorescence, immunohistochemistry and immunoblotting were utilized to elucidate the impact and mechanisms of PANO treatment on spermatogenesis. RNA-seq analysis was performed on mouse testes to elucidate the underlying mechanisms of PANO.

Results: The rates of sperm survival and movement were reduced while malformation rate of sperm increased in the 34.4-day PANO group. Gene Set Enrichment Analysis (GSEA) supported the significant role of PANO in modulating cilium movement, sperm axoneme assembly and flagellated sperm motility. Numbers of MVH⁺ cells were decreased while the numbers of SCP3⁺ cells were significantly increased after PANO treatment compared to those in the control (CTL) group. The protein levels of PLZF in PANO-treated testes were dramatically reduced along with the increased distribution changes of SOX9, F4/80 protein. Further data demonstrated that PANO impeded spermiogenesis at the stage XI in the 34.4-day PANO group and enhanced the transcriptome levels of histone variants H2bc4 and H1f2.

Conclusions: We have reported that PANO exerts a negative impact on the spermatogonial stem cell pool in mouse testes by disrupting its niche. This disruption leads to a reduction in germ cell numbers and impairs sperm function in mice, ultimately resulting in infertility. Moreover, PANO destabilizes the nucleosomes by increasing the transcriptional levels of H2bc4 and H1f2, affects the histone-to-protamine transition, and arrests spermiogenesis at the elongating spermatid stage. These findings also suggest that H2bc4 and H1f2 may be potential key biomarkers in the testis for diagnosing male infertility associated with aberrant histone hyperacetylation due to the exposure of environmental pollutants.

Fig. 1

The testicular damage caused by Panobinostat at various doses in mice. (A) Changes in body weight of mice treated with 7-day PANO after 8.6 days at different doses. (B) Pictures of the mouse testes treated with PANO after 8.6 days at different doses. C-E. The changes in the organ index of testis, unilateral testicular volume/weight and bilateral epididymal weight/weight when subjected to varying doses of PANO. F. H&E staining of testicular sections. The presence of asterisks indicated the non-functional seminiferous tubules. G. Statistical visualization of indicators linked to seminiferous tubule impairment. Scale bar: 50–20 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS = not significant compared to the CTL group

Fig. 2

Panobinostat impaired sperm function in mice. (A) Pictures of the mouse sperm in the 34.4-day CTL and PANO groups were taken. (B) The evaluation of sperm quality parameters in mice included the survival rate, movement rate, sperm density, and malformation rate in the 34.4-day CTL and PANO groups. (C) PCA was performed on the 8.6-day CTL and PANO groups in the mouse testes. (D) Statistical bar chart of the genes that were differently expressed between the 8.6-day CTL and PANO groups was illustrated. (E) Volcano plot of different expressed genes was performed on the 8.6-day CTL and PANO groups in the mouse testes. (F) Enrichment results for cilium movement, sperm axoneme assembly, and flagellated sperm motility were obtained via GSEA in the 8.6-day CTL and PANO groups. Scale bar: 25 μm. **P < 0.01; ***P < 0.001; ****P < 0.0001; NS = not significant compared to the CTL group

Fig. 3

The comparisons of pup numbers at different time points. (A) The mice were mated 8.6 days and 34.4 days after a 7 - day administration, and then their fertility was observed. (B) The comparisons of pup numbers at different time points. *P < 0.05

Fig. 4

Panobinostat damages the structure of the testes. (A) According to the development time of germ cells in mice, three-time-point models were established: 8.6-day groups, 34.4-day groups and 43-day groups. (B) The changes in body weight were observed in mice following exposure to PANO at different times. (C) Pictures of the mouse testes in CTL and PANO groups at different times. (D) The organ index of mouse testis was measured at 8.6, 34.4, and 43 days after undergoing a consecutive 7-day treatment. E-F. The seminiferous tubules of mice in the 34.4-day CTL and PANO groups were subjected to H&E staining and stereology analysis. The line segments represented the thickness of the seminiferous epithelium. Scale bar: 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 5

Panobinostat decreases the expression of HDAC genes in mouse testes. A-D. The gene expression of HDAC family in the CTL and PANO groups was detected by qRT-PCR. All quantitative data were presented as the mean ± standard error of the mean (SEM) and analyzed using the 2^(-ΔCt) method. E. The mRNA levels of HDACs were evaluated using the 2^(-ΔΔCt) methods. F. The protein expression of acH3K9 in the CTL and PANO groups was detected. G. The relative acH3K9 protein expression at different time points in the CTL and PANO groups was analyzed. All data were presented as the mean ± standard deviation of the mean (SD). The relative acH3K9 protein expression=(acH3K9/β-actin)/(Histone H3/β-actin) *P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant compared to the CTL group

Fig. 6

Panobinostat increases the numbers of SCP3⁺ cells in testes. A, C, E. Proteins were labeled by using immunofluorescence techniques to detect MVH, STRA8, and SCP3, respectively. B, D, F. Statistical comparisons of the numbers of MVH⁺, STRA8⁺, and SCP3⁺ cells between the CTL and 34.4-day PANO groups. All counting was in the positive seminiferous tubules. Scale bar: 50 μm. ****P < 0.0001; NS = not significant compared to the CTL group

Fig. 7

Panobinostat negatively affects spermatogonial stem cell pool by disrupting the microenvironmental niche in the testes. A-B. The PLZF protein was labeled by using immunofluorescence, and the quantification of PLZF-positive cells was performed. C. The RNA-seq data were analyzed to profile the expression of Leydig cells and Sertoli cells in the mouse testes. D-E. The SOX9 protein was labeled by using immunohistochemistry, and the quantification of SOX9-positive cells was performed. All counting was in the positive seminiferous tubules. F. The RNA-seq data were analyzed to profile the expression of macrophages in the mouse testes. G-H. The F4/80 protein was labeled by using immunohistochemistry, and the quantification of F4/80 protein was performed. **P < 0.01. ****P < 0.0001

Fig. 8

PANO increases the levels of H2bc4 and H1f2 in testes. (A) PAS staining result showed the spermatids in the 34.4-day PANO group were arrested in the step 11 of spermiogenesis. (B) TNP1 proteins were labeled by using immunofluorescence in the step 11–12. Scale bar: 50 μm. (C) The RNA-seq analysis of histone variants showed a significant increase in the transcript abundance of H2bc4 and H1f2 in the 8.6-day PANO group (q-value < 0.05 &|log₂FoldChange|>1.0), as compared to the CTL group