The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Abstract

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male's life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitro cell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2 was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.

Figure 1

Global level and co-immunofluorescence localization of H3K9me3 and ESET. Transcripts of Eset were examined by qRT-PCR in multiple tissue samples (a) and during postnatal development of the testis (b). Values were normalized to that of Gapdh. *P<0.05 compared with other tissues. (c) ESET expression and H3K9me3 level were examined by western blot during postnatal development of the testis. (d) Co-immunofluorescence localization of H3K9me3/ESET (red) and PLZF/KIT (green) in SSCs and differentiated spermatogonia. Bar=100 μm. White line boxes were shown at higher magnification

Figure 2

MACS and RNAi efficiency and SSC culture in vitro. (a) Immunofluorescence of Thy1 (red) and DAPI (blue) in presorted and two-step sorted cells. Bar=80 μm. (b) Proportion of Thy1-positive cells in presorted and two-step sorted fraction (n=4). More than 350 cells were counted for each sample. Asterisks indicate statistically significant differences (**P<0.01). (c) qRT-PCR analysis of Eset expression in the ESET-KD, NC and Mock SSCs group. Values were normalized to that of Gapdh, **P<0.01 compared with NC or Mock controls. (d) ESET and global H3K9me3 level were slightly decreased in the ESET-KD SSCs, examined by western blot. (e) Relative number of Lin28-positive cells in ESET-KD, NC and Mock SSC groups after 2 weeks culture. Five thousands of SSCs for each treatment were plated in multiple wells of a 96-well plate and cultured for 2 weeks (n=3). The number of Lin28-positive cells in ESET-KD group was lower than that in NC and Mock group (**P<0.01). (f–k) Culture of SSCs after transduced with lentiviral. ESET-KD SSCs (f and i), NC group (g and j) and Mock group (h and k) were analyzed after 2 weeks culture. Immunofluorescence for Lin28 (red) and DAPI (blue) were performed to count the number of SSCs (i–k). Bar=80 μm

Figure 3

Phenotypic characteristics of recipient testes transplanted with SSCs that were transduced with lentiviral particles. (a) Schematic diagram of the SSC transplantation strategy. (b) After injection, presence of dye in seminiferous tubules confirmed successful injection (left). After 10 weeks, GFP expression in seminiferous tubules indicates donor SSC-derived spermatogenesis (right). Bar=2 mm. (c) Testis from recipient mice transplanted with ESET-KD SSCs was smaller than that transplanted with NC SSCs. Bar=4 mm. (d) Testis weights in ESET-KD, NC and Mock group. The values were significantly lower in the ESET-KD group compared with the control groups (*P<0.05). (e) Hematoxylin-eosin staining of the testis tissue transplanted with ESET-KD and NC SSCs. Spermatogenesis was heavily impaired in ESET-KD group. Bar=100 μm. (f) Quantitative measurements of reconstitution of the seminiferous epithelium (n=3). More than five sections and an average of 80 tubules/section were counted for each sample. Asterisk indicates significant differences between the two groups (*P<0.05). (g) Western blot analysis of testis tissue transplanted with ESET-KD SSCs, NC SSCs and Mock SSCs

Figure 4

ESET regulated SSC apoptosis via caspase-dependent pathways. (a) TUNEL staining of SSC transduced with Eset-shRNA (ESET-KD), NC-shRNA (NC) or no lentiviral particles (Mock). Red color indicates TUNEL-positive cells. Counterstained with Hoechst 33342 are represented in blue. Bar=100 μm. (b) Graphical representation of the number of TUNEL-positive cells (n=4). More than 280 cells were counted for each sample. Asterisks indicate statistically significant differences (*P<0.05). (c) qRT-PCR analysis of gene expression in the ESET-KD, NC and Mock SSCs group. Values were normalized to that of Gapdh, *P<0.05 compared with NC or Mock controls. (d) Caspase-dependent apoptosis caused by lacking ESET examined by western blot. Caspase9 and the cleaved Caspase3 level were increased, and with the presence of cleaved PARP in ESET-KD SSCs

Figure 5

ESET regulated SSCs apoptosis by Cox4i2 through increasing H3K9me3 and DNA methylation. (a) Cross-linked ChIP assay was performed with IgG or anti-ESET IgG in C18-4 cells and direct binding of ESET on the promoter regions of Cox4i2, Sohlh2, Dazl, Nobox, Foxn1, Capase9 and Apaf1 was detected. *P<0.05 compared with IgG group. (b) ChIP assay was performed with H3K9me3 antibody in C18-4 cells transfected with Eset-siRNA, NC-siRNA or transfection reagent. Binding activity (relative to the input) of H3K9me3 on promoter regions of Cox4i2, Nobox, Dazl, Foxn1 and Sohlh2 was quantified. *P<0.05 compared with NC-siRNA or Mock controls. (c) Graphical representation of the number of TUNEL-positive cells (n=4). More than 256 cells were counted for each sample. Asterisks indicate statistically significant differences (**P<0.01). (d) Transcripts of Eset, Cox4i2 and Caspase9 were examined by qRT-PCR in C18-4 cells transfected with siRNA. (e) Bisulfite sequencing analysis of Cox4i2 (−0.5 kb) and Dazl promoter (−0.5 kb) in C18-4 cells. The position of CpG dinucleotide analyzed is indicated by a horizontal line. Open circles, unmethylated CpG; closed circles, methylated CpG

Figure 6

The schematic diagram of ESET regulation in SSCs. The schematic diagram demonstrates epigenetic regulation events in apoptosis process in spermatogonial stem/progenitor cells. ESET modulates H3K9me3, which represses the expression of Cox4i2 directly and also suppresses expression of Apaf1 via undetermined ways. Cox4i2 stimulates activation of Caspase9, which then cleaves downstream caspases (such as Caspase3) and PARP, leading to cell apoptosis