Abstract
Spermatogenesis is crucial for male fertility and is therefore tightly controlled by a variety of epigenetic regulators. However, the function of enhancer of zeste homolog 2 (EZH2) in spermatogenesis and the molecular mechanisms underlying its activity remain poorly defined. Here, we demonstrate that deleting EZH2 promoted spermatogonial differentiation and apoptosis. EZH2 is expressed in spermatogonia, spermatocytes and round and elongated spermatids from stage 9 to 11 but not in leptotene and zygotene spermatocytes. Knocking down Ezh2 in vitro using a lentivirus impaired self-renewal in spermatogonial stem cells (SSCs), and the conditional knockout of Ezh2 in spermatogonial progenitors promoted precocious spermatogonial differentiation. EZH2 functions to balance self-renewal and differentiation in spermatogonia by suppressing NEUROG3 and KIT via a direct interaction that is independent of its histone methyltransferase activity. Moreover, deleting Ezh2 enhanced the activation of CASP3 in spermatids, resulting in reduced spermatozoa production. Collectively, these data demonstrate that EZH2 plays a nonclassical role in the regulation of spermatogonial differentiation and apoptosis in murine spermatogenesis.
Introduction
Spermatogenesis is a highly organized and consistent process that requires a balance to be established between the self-renewal and differentiation of spermatogonial stem cells (SSCs). It is widely believed that SSCs, which are also known as Asingle (As) spermatogonia, are the most primitive spermatogonia and that they do not form intercellular bridges (Huckins 1971, Oakberg 1971, De Rooij & Russell 2000). Spermatogenesis starts with the decision to differentiate when a committed As spermatogonia undergoes mitosis to produce an Apaired (Apr) spermatogonia instead of self-renewing (De Rooij & Russell 2000). SSCs can also give rise to undifferentiated spermatogonial progenitors with different cellular states (Song & Wilkinson 2014). These spermatogonial progenitors include committed As, Apr and Aaligned (Aal) spermatogonia, which induce the full spermatogenesis process under normal conditions. However, under specific conditions, some spermatogonial progenitors (potential stem cells) can change modes from transient amplification to self-renewal and act as SSCs (Yoshida et al. 2007).
Undifferentiated spermatogonial (Aun), including As, Apr and Aal, are recognized by several protein markers, such as ZBTB16 (Costoya et al. 2004), LIN28A (Zheng et al. 2009), GFRA1 and NEUROG3 (NGN3). Two previous publications described the expression of NGN3 in germ cells (Yoshida et al. 2004) and provided convincing evidence that NGN3 is expressed in As, Apr and Aal spermatogonia, all of which eventually contribute to spermatogenesis in mature LacZ-labelled testis. Furthermore, the authors noted that NGN3 was not expressed in all As, Apr and Aal spermatogonia and was expressed at low or negative levels in actual stem cells and high levels in potential stem cells (Yoshida et al. 2007). NGN3+ germ cells have also been defined as spermatogonial progenitors that are committed to differentiation (Nakagawa et al. 2007, Song & Wilkinson 2014). Before meiosis is initiated, differentiating A1 spermatogonia go through a series of mitoses (into A2, A3, A4, Int and B types), and these differentiating spermatogonia are recognized by KIT (Yoshinaga et al. 1991). After spermatogonial amplification, meiosis is initiated under the control of retinoic acid (RA) (Hogarth & Griswold 2010), which regulates both STRA8 (Zhou et al. 2008) and KIT (Busada et al. 2015) during spermatogenesis.
In a recent study, germline cells obtained from neonatal testes were cultured in vitro, and the SSCs proliferated for a long period of time under specific culture conditions (Kanatsu-Shinohara et al. 2003, Zhang et al. 2012b). In this culture system, the SSCs colony consisted of a mixture of SSCs and spermatogonial progenitors, and the SSCs could not be recognized by their morphology or directly counted (Kanatsu-Shinohara et al. 2003, Zhang et al. 2012b). A widely accepted method for analysing real SSCs numbers in culture is to count the number of colonies that formed after a transplantation assay was performed (Brinster & Avarbock 1994, Brinster & Zimmermann 1994). In addition, there was no difference in the number of colonized sites after 1 and 4 months, but the lengths of the colonies increased from 0.73 to 5.78 mm (Nagano et al. 1999).
Previous studies have shown that EZH2 is a key factor during cancer progression, including ovarian cancer (Lin et al. 2016), prostate cancer (Yang & Yu 2013) and testicular germ-cell tumours (Hinz et al. 2010), indicating that EZH2 plays a specific role in germ-cell progression. Furthermore, an increasing amount of evidence suggests that EZH2 is a key regulator of stem cell maintenance and differentiation during ontogenesis in processes including natural killer cell differentiation (Yin et al. 2015) and adult hippocampal neurogenesis (Zhang et al. 2014). In addition to its classical function of trimethylating histone H3K27, EZH2 also methylates non-histone targets, such as GATA4 at Lys 299 (He et al. 2012) and regulates oocyte maturation by directly binding to BUBR1 independent of its histone methyltransferase activity (Qu et al. 2016). EZH2 is expressed at high levels in adult testis (Lambrot et al. 2012), suggesting that it plays a regulatory role in spermatogenesis. In this study, we explored the localization of EZH2 in male germ cells and investigated its function in the regulation of spermatogenesis.
Materials and methods
Mouse lines and crosses
The animal experiments were approved by the Committee on Animal Care of the Institute of Zoology, Chinese Academy of Sciences. The Ezh2flox/flox mice were generated by Prof. Yang Xiao (Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China), and the Nuerog3-cre mice (006333 – B6.FVB(Cg)-Tg(Neurog3-cre)C1Able/J) were obtained from the Jackson Laboratory (Schonhoff et al. 2004). All of the mice were bred in a C57BL/6 background. Ezh2flox/flox mice were mated with transgenic mice carrying Neurog3-cre to generate Ezh2-/flox;Neurog3-cre mice (KO mice). DNA was isolated from tails and genotyped using PCR.
Fertility valuation
The Ezh2flox/flox and Ezh2 KO mice were mated with C57BL/6 female mice, and vaginal plugs were checked every morning. Female mice with a vaginal plug were removed and replaced with new C57BL/6 female mice. The average litter size of each group was calculated from ten nests.
Sperm counts and motility analysis
Total sperm counts were obtained according to Roy et al. (2007). Briefly, epididymal caput and cauda were minced and incubated in prewarmed M16 medium (Sigma-Aldrich) at 37°C in air containing 5% CO2 for 30 min to allow the sperm to swim out. Then, the sperm were diluted in water and counted using a haemocytometer. To assess sperm motility, only the caudal spermatozoa were diluted in M16 medium to a density of 106/mL. They were then transferred to a MicroCell fixed-depth chamber (Conception Technologies, India) on a 37°C stage, and one hundred sperm were scored as motile or immotile. For each sample, sperm counts and motility values were obtained twice and averaged.
Immunohistochemistry and immunofluorescence
Mouse testes fixed in 4% paraformaldehyde at 4°C overnight were dehydrated, embedded in paraffin and cut into 5 µm thick sections. Rehydrated sections were subjected to antigen retrieval, blocked in 5% BSA (0.1% Triton X-100 in 5% BSA) and incubated with primary antibody at 4°C overnight. After incubation in 3% H2O2 for 15 min, the section was incubated with secondary and tertiary antibodies at 37°C for 1 h each, followed by DAB staining (Zhong Shan Technology, Beijing, China), hematoxylin staining, dehydration and mounting with neutral gum. For immunofluorescence, after incubation with primary antibody, sections were incubated with secondary antibody at 37°C for 1 h and then stained with DAPI (Sigma) for 15 min. After the sections were mounted with anti-quencher fluorescence decay, images were captured using a Zeiss 780 laser scanning confocal microscope.
Real-time RT-PCR
Testicular total RNA was extracted using TRIzol reagent (Invitrogen). RNA integrity measurement and cDNA synthesis were performed as described (Barrionuevo et al. 2009). Data were normalized according to Gapdh, and the relative gene expression level was calculated according to the ΔΔCt method (Livak & Schmittgen 2001). The primer sequences are listed in Supplementary Table 1 (see section on supplementary data given at the end of this article).
Western blotting
Testes were lysed in RIPA lysis buffer (Biomed, Shanghai) containing protease inhibitor cocktail tablets (Roche) and PMSF. After electrophoresis on 10% SDS-PAGE gels, proteins were transferred to nitrocellulose membranes and blocked in 5% skimmed milk. The membranes were incubated with primary antibody at 4°C overnight and horseradish peroxidase-labelled secondary antibody for 1 h at 37°C subsequently. Images were captured using ECL Western Blotting Substrate (Thermo Scientific Pierce).
BrdU incorporation
The control and KO mice were intraperitoneally injected with 50 mg/kg BrdU (Sigma) and were killed two hours later to identify BrdU-positive adult-born cells.
Co-immunoprecipitation
Testes were collected to perform co-immunoprecipitation experiments according to the instructions of the Pierce co-immunoprecipitation Kit (Thermo Scientific). The proteins obtained in co-immunoprecipitation assays were subjected to Western blotting and analysed using specific antibodies.
Cell culture
Spermatogonia were isolated, enriched and cultured according to previously described procedures (Kanatsu-Shinohara et al. 2003, Zhang et al. 2012b). Briefly, a testicular cell suspension of 5–6-dpp mouse pups (progenies of C57BL6 and DBA/2) was obtained using a two-step enzymatic digestion protocol with 1 mg/mL of type-IV collagenase (Sigma) and 0.25% trypsin (Invitrogen). Both digestions required 500 μg/mL DNase I (Sigma) to protect against DNA degradation. The dissociated cells were centrifuged, resuspended in DPBS containing 1% FBS (HyClone, USA), and then centrifuged again in a 30% Percoll solution. Then, the spermatogonia were enriched using MACS to isolate Thy-1+ cells. Spermatogonia were cultured on MEF treated with Mitomycin C in serum-free MEMα (Invitrogen) supplemented with other components, as previously described (Kanatsu-Shinohara et al. 2003).
RNA interference of Ezh2 by shRNA
The target sequences are listed in Supplementary Table 2. The lentiviruses were packaged according to the instructions provided in the Lenti-Easy Packaging System (Genechem). The titers of the viral particle-containing supernatants were measured using the fluorescent counting method according to the instructions mentioned above and ranged from 2 × 106 to 5 × 106 IU/mL. The multiplicity of infection (MOI) is the ratio of the number of viral particles to the number of target cells when approximately 80% of target cells are transfected. The MOI in our experiment was 13. The viral particles were then introduced to the cultured spermatogonia.
Flow cytometric analysis
The cell suspensions were fixed in 1% paraformaldehyde at RT for 15 min and then permeabilized (0.1% Triton X-100 in PBS) for 20 min, washed with DPBS twice, blocked in 0.5% BSA for 30 min at RT and incubated with primary antibodies at 4°C overnight. After the cells were washed in DPBS, they were incubated in secondary antibodies for 1 h at RT and then washed and filtered using a cell strainer with a 40 μm pore size (Becton Dickinson, USA). They were then resuspended in DPBS containing 1% FBS (HyClone) and subjected to flow cytometry. The results were analysed using a FACS-Calibur system (BD Biosciences, USA). To analyse DNA ploidy, the cells were fixed in 70% ethanol at 4°C overnight and then washed in DPBS. Then, the cells were incubated with the staining solution (0.02 mg/mL propidium iodide and 0.2 mg/mL RNase A) at 37°C for 20 min and filtered before being subjected to flow cytometry. The results were analysed using a FACS-Calibur system (BD Biosciences).
Antibodies
The antibodies used in this study are listed in the Supplementary Table 3.
Statistical analysis
All statistical analyses were performed using SPSS, version 16.0. All experiments were repeated at least three times, and data for evaluated parameters are reported as means ± s.e.m.
Results
Expression pattern of EZH2 in male germ cells
To analyse the expression pattern of EZH2 in spermatogonia, testes were obtained from 10-day-old mice, in which the most advanced germ cells are type B spermatogonial. The tissues were co-stained for EZH2, DDX4 and LIN28A. The results showed that EZH2 was localized on the nucleus of spermatogonial (DDX4+) and that immunolabelling was stronger in undifferentiated (DDX4+, LIN28A+) than in differentiating (DDX4+, LIN28A−) spermatogonia (Fig. 1A). These results suggest that EZH2 plays a potential role in maintaining an undifferentiated spermatogonial state. The expression pattern of EZH2 was also analysed in spermatocytes obtained from adult testis. Different stages of primary spermatocytes were distinguished by the expression of SYCP3 (Yuan et al. 2000, de la Fuente et al. 2007, Abby et al. 2016). Immunostaining for EZH2 was weak in preleptotene, rarely detected in leptotene and zygotene, apparent in early pachytene and strong in late pachytene and diplotene spermatocytes (Fig. 1B). Labelling persisted in round spermatids but became weaker as the spermatids elongated during progression from stage 9 to 11 and then disappeared by stage 12 (Fig. 1C), indicating that EZH2 performs a specific function during the later phases of meiosis. An overview of the expression of EZH2 showed that the strongest signals were observed in late pachytene and diplotene spermatocytes, stronger signals were observed in round spermatids and undifferentiated spermatogonial, and weak signals were observed in other germ cells (Fig. 1D).
Expression pattern of EZH2 in male germ cells. (A) Immunofluorescence staining of EZH2 with DDX4 and LIN28A in the testis of 10-day-old mice. (B) Immunofluorescence staining of EZH2 with SYCP3 in preleptotene (a), leptotene (b), zygotene (c), early pachytene (d), late pachytene (e) and diplotene (f) of adult testis. (C) Immunofluorescence staining of EZH2 in spermatid. (D) An overall view of EZH2 in adult testis. Scale bars in (A): 20 µm, (B): 2 µm, (C): 10 µm, (D): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Expression pattern of EZH2 in male germ cells. (A) Immunofluorescence staining of EZH2 with DDX4 and LIN28A in the testis of 10-day-old mice. (B) Immunofluorescence staining of EZH2 with SYCP3 in preleptotene (a), leptotene (b), zygotene (c), early pachytene (d), late pachytene (e) and diplotene (f) of adult testis. (C) Immunofluorescence staining of EZH2 in spermatid. (D) An overall view of EZH2 in adult testis. Scale bars in (A): 20 µm, (B): 2 µm, (C): 10 µm, (D): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Expression pattern of EZH2 in male germ cells. (A) Immunofluorescence staining of EZH2 with DDX4 and LIN28A in the testis of 10-day-old mice. (B) Immunofluorescence staining of EZH2 with SYCP3 in preleptotene (a), leptotene (b), zygotene (c), early pachytene (d), late pachytene (e) and diplotene (f) of adult testis. (C) Immunofluorescence staining of EZH2 in spermatid. (D) An overall view of EZH2 in adult testis. Scale bars in (A): 20 µm, (B): 2 µm, (C): 10 µm, (D): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Ezh2 conditional knockout mice have fewer sperm
To determine whether Ezh2 regulates spermatogenesis, we generated Ezh2 conditional knockout mice. In Ezh2flox/flox mice (Fig. 2A), the fourth exon is floxed, and in Nuerog3-cre mice, cre activity is initiated in Neurog3-positive spermatogonial progenitors. Ezh2flox/−;Neurog3-cre mice (KO mice) were obtained by crossing Ezh2flox/flox mice with Neurog3-cre mice. The knockout efficiency of EZH2 in the KO mice testes was confirmed using immunostaining, Western blotting and real-time RT-PCR, which revealed that both the mRNA and the protein levels of EZH2 were significantly reduced (Fig. 2B, C and D). Moreover, EZH2 deletion in the KO mice also significantly reduced H3K27me3 (Fig. 2D and E). A histological analysis showed that the 6-month-old KO mice had normal testes (Fig. 3A and B) and released spermatozoa into the lumen during stages VII–VIII of the seminiferous epithelial cycle (Fig. 3D). However, the average litter size was significantly lower after 4 months in the KO than in the WT litters (Fig. 3C). Furthermore, the results of H&E staining of the cauda epididymidis and sperm cell counting in the epididymis at different ages showed that sperm numbers were lower in the KO mice and that the difference became more and more significant with the age (Fig. 3E and F). In addition, sperm motility was normal in the epididymis in 6-month-old KO mice (Fig. 3G), suggesting that the reduction in average litter size was caused by a lower sperm number and not reduced sperm motility.
Ezh2 conditional knockout mice. (A) The Ezh2flox/flox mice floxed the fourth exon. (B) Immunofluorescence staining of EZH2 in the KO mice testes. (C) The real-time RT-PCR of Ezh2 in KO mice testes. (D) Western blotting of EZH2 and H3K27me3 in KO mice testes. (E) Immunohistochemistry staining of H3K27me3 and IgG in KO mice testes. Scale bars in (B): 100 µm, (E): 20 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Ezh2 conditional knockout mice. (A) The Ezh2flox/flox mice floxed the fourth exon. (B) Immunofluorescence staining of EZH2 in the KO mice testes. (C) The real-time RT-PCR of Ezh2 in KO mice testes. (D) Western blotting of EZH2 and H3K27me3 in KO mice testes. (E) Immunohistochemistry staining of H3K27me3 and IgG in KO mice testes. Scale bars in (B): 100 µm, (E): 20 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Ezh2 conditional knockout mice. (A) The Ezh2flox/flox mice floxed the fourth exon. (B) Immunofluorescence staining of EZH2 in the KO mice testes. (C) The real-time RT-PCR of Ezh2 in KO mice testes. (D) Western blotting of EZH2 and H3K27me3 in KO mice testes. (E) Immunohistochemistry staining of H3K27me3 and IgG in KO mice testes. Scale bars in (B): 100 µm, (E): 20 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Ezh2 conditional knockout mice have fewer sperm. (A) Testes of 6-month-old KO and control mice. (B) Testes weight of KO and control mice at different ages. (C) Average litter size of KO and control mice at different age (N = 10). H&E staining of the (D) testes and the (E) cauda epididymidis at different ages. (F) The sperm number in epididymis of KO and control mice at different ages. (G) The sperm motility of 6-month-old KO and control mice. Scale bars in (A): 2 mm, (D): 100 µm, (E): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Ezh2 conditional knockout mice have fewer sperm. (A) Testes of 6-month-old KO and control mice. (B) Testes weight of KO and control mice at different ages. (C) Average litter size of KO and control mice at different age (N = 10). H&E staining of the (D) testes and the (E) cauda epididymidis at different ages. (F) The sperm number in epididymis of KO and control mice at different ages. (G) The sperm motility of 6-month-old KO and control mice. Scale bars in (A): 2 mm, (D): 100 µm, (E): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Ezh2 conditional knockout mice have fewer sperm. (A) Testes of 6-month-old KO and control mice. (B) Testes weight of KO and control mice at different ages. (C) Average litter size of KO and control mice at different age (N = 10). H&E staining of the (D) testes and the (E) cauda epididymidis at different ages. (F) The sperm number in epididymis of KO and control mice at different ages. (G) The sperm motility of 6-month-old KO and control mice. Scale bars in (A): 2 mm, (D): 100 µm, (E): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency elevates apoptosis
To determine whether the reduction in spermatids was caused by aberrant apoptosis, we performed immunofluorescence staining of cleaved CASP3 (CAS3), which is the large fragment of activated CASP3. Compared to the cytoplasmic expression of CAS3 in the mature spermatids of control mice, CAS3 was aberrantly expressed in the nucleus of elongated spermatids in the KO mice (Fig. 4A), and the protein level of CAS3 was increased in KO testes (Fig. 4C). These data suggest that EZH2 suppresses apoptosis by inhibiting CASP3 activation in spermatids. Moreover, the mRNA and protein levels of Ddx4 and Acr in the testis was significantly lower in the KO mice (Fig. 4B and C), indicating a reduction in germ-cell and spermatid numbers. However, the protein level of SYCP3 (Fig. 4B and C) was higher in the KO mice, indicating the observed reduction in germ cells was induced by apoptosis during a later stage of meiosis.
EZH2 deficiency elevates apoptosis. (A) Immunofluorescence staining of the cleaved CASP3 (CAS3) in the KO and control mice. (B) Real-time RT-PCR and (C) Western blotting of key marker genes in the testes of KO and control mice. Scale bars in (A): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency elevates apoptosis. (A) Immunofluorescence staining of the cleaved CASP3 (CAS3) in the KO and control mice. (B) Real-time RT-PCR and (C) Western blotting of key marker genes in the testes of KO and control mice. Scale bars in (A): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency elevates apoptosis. (A) Immunofluorescence staining of the cleaved CASP3 (CAS3) in the KO and control mice. (B) Real-time RT-PCR and (C) Western blotting of key marker genes in the testes of KO and control mice. Scale bars in (A): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
In addition, the mRNA expression levels of Id4, which is specifically expressed in SSCs (Chan et al. 2014, Sun et al. 2015), and Ret, which is important for SSCs self-renewal (Naughton et al. 2006), were significantly lower in the KO mice (Fig. 4B), suggesting a defect in SSCs self-renewal. Moreover, the protein levels of ZBTB16 and STRA8 were higher in the KO mice (Fig. 4C), indicating an increase in spermatogonial progenitors and the initiation of meiosis in KO mouse testes. Finally, the protein levels of NGN3 and KIT were higher (Fig. 4C) in the KO mouse testes, suggesting that the proportion of differentiating spermatogonial was also increased.
EZH2 deficiency impairs SSCs self-renewal and promotes spermatogonial differentiation
To determine whether Ezh2 deletion promoted spermatogonial differentiation, we performed immunofluorescence staining for KIT, LIN28A and ZBTB16. Consistent with the observed increase in the protein level of KIT, the KO mice expressed a high level of KIT, which was aberrantly expressed in some of the undifferentiated spermatogonial (PLZF+, LIN28A+) in the KO mice (Fig. 5A). The aberrant presence of KIT indicates the precocious transition of spermatogonia from an undifferentiated to a differentiated state. Moreover, following the intraperitoneal injection of BrdU, more BrdU-positive signals were observed in stage VII–VIII seminiferous epithelial cells in the KO mice than in the controls (Fig. 5B and C). In addition, fluorescent flow cytometry analysis of total testicular cell suspensions also showed that the number of BrdU-positive cells was higher in the KO mice (Fig. 5D), indicating enhanced DNA synthesis and proliferation in spermatogonia prior to the initiation of meiosis. Moreover, flow cytometric analysis of total testicular cell suspensions showed that the proportions of 2 N and 4 N ploidy were higher, while the proportion of 1 N ploidy was lower in the KO mice (Fig. 5E). These data also suggest an elevated rate of proliferation in spermatogonia in addition to impaired spermatogenesis. The proliferation of spermatogonia may have been increased in the KO mice as a result of the promotion of self-renewal of SSCs, enhanced differentiation in undifferentiated spermatogonia or both.
EZH2 deficiency promotes spermatogonial differentiation. (A) Immunofluorescence staining of KIT with LIN28A and ZBTB16 in the KO and control mice testes and the white arrow indicates ZBTB16+ and LIN28A+ undifferentiated spermatogonia. (B) Immunofluorescence staining of BrdU in the KO and control mice testes two hours after the BrdU incorporation assay. (C) Quantitative analysis of the BrdU-positive cell number in the KO and control mice testes. (D) Fluorescence flow cytometry analysis of the BrdU-positive cell numbers in total testicular cell suspension in the KO and control mice. (E) DNA ploidy analysis of the total testicular cell suspension in the KO and control mice. Scale bars in (A): 50 µm, (B): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency promotes spermatogonial differentiation. (A) Immunofluorescence staining of KIT with LIN28A and ZBTB16 in the KO and control mice testes and the white arrow indicates ZBTB16+ and LIN28A+ undifferentiated spermatogonia. (B) Immunofluorescence staining of BrdU in the KO and control mice testes two hours after the BrdU incorporation assay. (C) Quantitative analysis of the BrdU-positive cell number in the KO and control mice testes. (D) Fluorescence flow cytometry analysis of the BrdU-positive cell numbers in total testicular cell suspension in the KO and control mice. (E) DNA ploidy analysis of the total testicular cell suspension in the KO and control mice. Scale bars in (A): 50 µm, (B): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency promotes spermatogonial differentiation. (A) Immunofluorescence staining of KIT with LIN28A and ZBTB16 in the KO and control mice testes and the white arrow indicates ZBTB16+ and LIN28A+ undifferentiated spermatogonia. (B) Immunofluorescence staining of BrdU in the KO and control mice testes two hours after the BrdU incorporation assay. (C) Quantitative analysis of the BrdU-positive cell number in the KO and control mice testes. (D) Fluorescence flow cytometry analysis of the BrdU-positive cell numbers in total testicular cell suspension in the KO and control mice. (E) DNA ploidy analysis of the total testicular cell suspension in the KO and control mice. Scale bars in (A): 50 µm, (B): 100 µm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
To determine the function of EZH2 in SSCs self-renewal, an in vitro assay was performed using a lentivirus RNAi against Ezh2 in SSCs. Immunostaining performed in cultured cells (Fig. 6A) showed that EZH2 was expressed in GFRA1+ SSCs but not MEF cells (white arrow). The GFRA1+ SSCs that were cultured in the maintenance system were transfected with sh-Ezh2 viral particles (RNAi group) or sh-scramble viral particles (control group). Then, we used flow cytometry sorting to determine which cells were transfected with different lentiviral particles, and the same number of SSCs were screened in each group. The SSCs were cultured for one week, and in the RNAi group, the knockdown efficiency of Ezh2 was approximately 50% at the mRNA (Fig. 6C) and protein (Fig. 6D) levels. Moreover, the colonies that formed in the culture system were smaller in the RNAi group (Fig. 6B). The same numbers of cultured spermatogonia were selected from the RNAi and control groups and transplanted into the testes of busulfan-treated mice. The results showed that there were fewer GFP+ seminiferous epithelia and significantly fewer colonies in the RNAi than in the control tested (Fig. 6E and F). These data indicate that the self-renewal is defective in the Ezh2 knockdown SSCs.
EZH2 deficiency impairs SSCs self-renewal. (A) Immunofluorescence staining of EZH2 with GFRA1 in the cultured cells. The white arrow indicates the MEF cells. (B) The cultured colony formed in the RNAi and control groups before transplantation. (C) Real-time RT-PCR and (D) Western blotting of EZH2 and several key marker genes in the RNAi and control groups before transplantation. (E) Recipient testes of the RNAi and control groups one month after the transplantation assay. (F) Quantity valuation of the colony number in the seminiferous tubules from RNAi and control groups. Scale bars in (A): 20 µm, (B): 50 µm, (E): 1 mm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency impairs SSCs self-renewal. (A) Immunofluorescence staining of EZH2 with GFRA1 in the cultured cells. The white arrow indicates the MEF cells. (B) The cultured colony formed in the RNAi and control groups before transplantation. (C) Real-time RT-PCR and (D) Western blotting of EZH2 and several key marker genes in the RNAi and control groups before transplantation. (E) Recipient testes of the RNAi and control groups one month after the transplantation assay. (F) Quantity valuation of the colony number in the seminiferous tubules from RNAi and control groups. Scale bars in (A): 20 µm, (B): 50 µm, (E): 1 mm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 deficiency impairs SSCs self-renewal. (A) Immunofluorescence staining of EZH2 with GFRA1 in the cultured cells. The white arrow indicates the MEF cells. (B) The cultured colony formed in the RNAi and control groups before transplantation. (C) Real-time RT-PCR and (D) Western blotting of EZH2 and several key marker genes in the RNAi and control groups before transplantation. (E) Recipient testes of the RNAi and control groups one month after the transplantation assay. (F) Quantity valuation of the colony number in the seminiferous tubules from RNAi and control groups. Scale bars in (A): 20 µm, (B): 50 µm, (E): 1 mm.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
In addition, consistent with the in vivo results, the mRNA expression level of Id4 was also significantly reduced (Fig. 6C) in the RNAi group, while the expression of NGN3, STRA8 and KIT were increased at both the mRNA and protein levels (Fig. 6C and D), confirming that spermatogonial differentiation was promoted when Ezh2 was knocked down in SSCs.
Together, these data indicate that EZH2 deficiency impairs SSCs self-renewal and promotes spermatogonial differentiation.
EZH2 functions by directly binding to NGN3 and KIT
In both the knockout and knockdown models of Ezh2, two protein levels of two key regulators of spermatogonial differentiation, NGN3 and KIT, were significantly higher. To determine whether EZH2 inhibits spermatogonial differentiation by directly regulating these two proteins, a co-immunoprecipitation assay was performed, and the results showed that EZH2 pulled-down NGN3 and KIT and vice versa (Fig. 7A, B, C and D), indicating that EZH2 can inhibit NGN3 and KIT via a direct interaction. Based on these data, we propose a model in which EZH2 regulates spermatogonial differentiation by maintaining spermatogonia in an undifferentiated state by inhibiting NGN3 and KIT expression. When EZH2 is deficient, NGN3 and KIT accumulate in spermatogonia, and this in turn promotes spermatogonial differentiation (Fig. 7F).
EZH2 functions by directly binding to NGN3 and KIT. (A) The co-immunoprecipitation of EZH2 with NGN3. (B) The co-immunoprecipitation of NGN3 with EZH2. (C) The co-immunoprecipitation of EZH2 with KIT. (D) The co-immunoprecipitation of KIT with EZH2. (E) The Asingle model and several genes expressed during spermatogenesis summarized from introductions. (F) Model of the nonclassical function of EZH2 in the regulation of spermatogonial differentiation.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 functions by directly binding to NGN3 and KIT. (A) The co-immunoprecipitation of EZH2 with NGN3. (B) The co-immunoprecipitation of NGN3 with EZH2. (C) The co-immunoprecipitation of EZH2 with KIT. (D) The co-immunoprecipitation of KIT with EZH2. (E) The Asingle model and several genes expressed during spermatogenesis summarized from introductions. (F) Model of the nonclassical function of EZH2 in the regulation of spermatogonial differentiation.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
EZH2 functions by directly binding to NGN3 and KIT. (A) The co-immunoprecipitation of EZH2 with NGN3. (B) The co-immunoprecipitation of NGN3 with EZH2. (C) The co-immunoprecipitation of EZH2 with KIT. (D) The co-immunoprecipitation of KIT with EZH2. (E) The Asingle model and several genes expressed during spermatogenesis summarized from introductions. (F) Model of the nonclassical function of EZH2 in the regulation of spermatogonial differentiation.
Citation: Reproduction 154, 5; 10.1530/REP-17-0302
Discussion
EZH2 immunofluorescence was previously detected only in the nuclear apical region in round spermatids of murine testis (Lambrot et al. 2012). This is different from our observations. However, the author also mentioned that EZH2 was detected from PND6 to PND14 using Western blot analysis, indicating that EZH2 is present from spermatogonia to spermatocytes. The author explained that this result may be because immunofluorescence is less sensitive than Western blotting (Lambrot et al. 2012). However, we bought the polyclonal EZH2 antibody with the same item number from Abcam, and the instructions included state that this antibody should not be used for immunofluorescence in paraffin sections but can be used for immunohistochemical staining. This is potentially why the results of immunofluorescence staining for EZH2 that were previously reported are not consistent with the results of Western blotting but similar to staining for IgG (Supplementary Fig. 3). These results indicate that the signal is nonspecific.
The expression pattern of EZH2 in male germ cells was validated using three different antibodies containing synthetic peptide derived from residues 50 to 150 (Abcam), 156 to 256 (BD) or 304 to 404 (CST) of human EZH2. Immunostaining for EZH2 and IgG (negative control) showed that the three different peptides for EZH2 detected similar signals in germ cells; however, no specific signals were detected in somatic cells (Supplementary Figs 1 and 2), which was similar to the results of immunostaining for IgG (Supplementary Figs 4 and 5). Hence, the weak signals that the CST antibody detected in somatic cells are likely nonspecific background signal.
The role of EZH2 in suppressing adult pluripotent stem cell differentiation has also been reported in retinal progenitors, in which EZH2 maintains the proliferation of retinal progenitors by inhibiting the expression of developmental regulators and thereby regulating the timing of late differentiation (Zhang et al. 2015). The reduction of SSCs self-renewal was confirmed using in vitro knockdown assays, and the increase in the proportion of differentiating spermatogonia and primary spermatocytes in the KO mouse testes indicated that EZH2 plays a role in regulating the balance between SSCs self-renewal and differentiation. Moreover, a previous study reported that inducing the deletion of Ezh2 in neural stem cells/progenitor cells resulted in fewer neurons (Zhang et al. 2014), similar to the effect on spermatids in our KO mice model. The induction of apoptosis by EZH2 deficiency has also been reported in tumours (Zhang et al. 2012a, Liu et al. 2015, Yu et al. 2016), and the activation of CASP3 by EZH2 may be mediated by a specific microRNA (Wang et al. 2014, Liu et al. 2015, Yu et al. 2016). Whether the enhanced CASP3 activation observed in KO mouse testes was induced by aberrant microRNA expression needs to be further explored.
Histone modification is vital for normal spermatogenesis (Okada et al. 2007, Tachibana et al. 2007, Kolthur-Seetharam et al. 2009), and during this process, genes are regulated by the deposition of H3K27me3. In this study, we found that Neurog3 and Zbtb16 were significantly upregulated at the mRNA level when EZH2 was deficient, suggesting that these two genes might be regulated by EZH2 methyltransferase activity. However, we found that H3K27me3 was reduced but not absent in germ cells, indicating that other histone methyltransferases, such as EZH1, are compensating for EZH2. Previous reports have shown that EZH1 can compensate for the loss of EZH2 in H3K27me3 deposition in a variety of tissues (Shen et al. 2008, Bardot et al. 2013, Lui et al. 2016, Zhu et al. 2016, Mu et al. 2017). It would therefore be useful to evaluate the role of H3K27me3 deposition in the regulation of spermatogenesis using EZH1/2 single- and double-knockout mice models and to explore this regulatory network using Chip-seq analysis. The data presented in this manuscript are focused on non-canonical function of EZH2 that have been previously reported in oocyte meiotic maturation (Qu et al. 2016), in which key regulatory protein was found to directly combine with EZH2.
In conclusion, our findings indicate that EZH2 is a key regulator of spermatogonial differentiation and apoptosis, and these data provide new insights into the molecular mechanisms underlying the conservation of male fertility.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-17-0302.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants from Major Research Plan ‘973’ Project (2012CB944702, LNCCC-D50-2015, LNCCC-bib9-2015), National Natural Science Foundation of China (31501161, 31501953, 31501198, 31471352, 31471400, 81270662 and 31171380), Changsha Academician Workstation and other Academician Workstation (Shenyang, Shandong) Support.
Acknowledgements
The authors thank Prof. Yang Xiao (Academy of Military Medical Sciences) for providing the Ezh2flox/flox mice.
References
Abby E, Tourpin S, Ribeiro J, Daniel K, Messiaen S, Moison D, Guerquin J, Gaillard JC, Armengaud J & Langa F et al. 2016 Implementation of meiosis prophase I programme requires a conserved retinoid-independent stabilizer of meiotic transcripts. Nature Communications 7. (doi:10.1038/ncomms10324)
Bardot ES, Valdes VJ, Zhang J, Perdigoto CN, Nicolis S, Hearn SA, Silva JM & Ezhkova E 2013 Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells. Embo Journal 32 1990–2000. (doi:10.1038/emboj.2013.110)
Barrionuevo F, Georg I, Scherthan H, Lecureuil C, Guillou F, Wegner M & Scherer G 2009 Testis cord differentiation after the sex determination stage is independent of Sox9 but fails in the combined absence of Sox9 and Sox8. Developmental Biology 327 301–312. (doi:10.1016/j.ydbio.2008.12.011)
Brinster RL & Avarbock MR 1994 Germline transmission of donor haplotype following spermatogonial transplantation. PNAS 91 11303–11307. (doi:10.1073/pnas.91.24.11303)
Brinster RL & Zimmermann JW 1994 Spermatogenesis following male germ-cell transplantation. PNAS 91 11298–11302. (doi:10.1073/pnas.91.24.11298)
Busada JT, Chappell VA, Niedenberger BA, Kaye EP, Keiper BD, Hogarth CA & Geyer CB 2015 Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse. Developmental Biology 397 140–149. (doi:10.1016/j.ydbio.2014.10.020)
Chan F, Oatley MJ, Kaucher AV, Yang Q-E, Bieberich CJ, Shashikant CS & Oatley JM 2014 Functional and molecular features of the Id4(+) germline stem cell population in mouse testes. Genes and Development 28 1351–1362. (doi:10.1101/gad.240465.114)
Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ & Pandolfi PP 2004 Essential role of Plzf in maintenance of spermatogonial stem cells. Nature Genetics 36 653–659. (doi:10.1038/ng1367)
de la Fuente R, Parra MT, Viera A, Calvente A, Gomez R, Suja JA, Rufas JS & Page J 2007 Meiotic pairing and segregation of achiasmate sex chromosomes in eutherian mammals: The role of SYCP3 protein. PLoS Genetics 3 2122–2134. (doi:10.1371/journal.pgen.0030198)
De Rooij DG & Russell LD 2000 All you wanted to know about spermatogonia but were afraid to ask. Journal of Andrology 21 776–798. (doi:10.1002/j.1939-4640.2000.tb03408.x)
He AB, Shen XH, Ma Q, Cao JJ, von Gise A, Zhou PZ, Wang G, Marquez VE, Orkin SH & Pu WT 2012 PRC2 directly methylates GATA4 and represses its transcriptional activity. Genes and Development 26 37–42. (doi:10.1101/gad.173930.111)
Hinz S, Magheli A, Weikert S, Schulze W, Krause H, Schrader M, Miller K & Kempkensteffen C 2010 Deregulation of EZH2 expression in human spermatogenic disorders and testicular germ cell tumors. World Journal of Urology 28 631–635. (doi:10.1007/s00345-009-0498-6)
Hogarth CA & Griswold MD 2010 The key role of vitamin A in spermatogenesis. Journal of Clinical Investigation 120 956–962. (doi:10.1172/JCI41303)
Huckins C 1971 Spermatogonial stem cell population in adult rats. 1. Their morphology, proliferation and maturation. Anatomical Record 169 533. (doi:10.1002/ar.1091690306)
Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S & Shinohara T 2003 Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biology of Reproduction 69 612–616. (doi:10.1095/biolreprod.103.017012)
Kolthur-Seetharam U, Teerds K, de Rooij DG, Wendling O, McBurney M, Sassone-Corsi P & Davidson I 2009 The histone deacetylase SIRT1 controls male fertility in mice through regulation of hypothalamic-pituitary gonadotropin signaling. Biology of Reproduction 80 384–391. (doi:10.1095/biolreprod.108.070193)
Lambrot R, Jones S, Saint-Phar S & Kimmins S 2012 Specialized distribution of the histone methyltransferase Ezh2 in the nuclear apical region of round spermatids and its interaction with the histone variant H1t2. Journal of Andrology 33 1058–1066. (doi:10.2164/jandrol.111.013870)
Lin L, Wang Z, Jin H, Shi H, Lu Z & Qi Z 2016 MiR-212/132 is epigenetically downregulated by SOX4/EZH2-H3K27me3 feedback loop in ovarian cancer cells. Tumour Biology 37 15719–15727. (doi:10.1007/s13277-016-5339-9)
Liu FH, He YK, Shu RH & Wang SK 2015 MicroRNA-1297 regulates hepatocellular carcinoma cell proliferation and apoptosis by targeting EZH2. International Journal of Clinical and Experimental Pathology 8 4972–4980.
Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25 402–408. (doi:10.1006/meth.2001.1262)
Lui JC, Garrison P, Nguyen Q, Ad M, Keembiyehetty C, Chen W, Jee YH, Landman E, Nilsson O & Barnes KM et al. 2016 EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy. Nature Communications 7. (doi:10.1038/ncomms13685)
Mu W, Starmer J, Shibata Y, Yee D & Magnuson T 2017 EZH1 in Germ Cells Safeguards the Function of PRC2 during Spermatogenesis. Developmental Biology 424 198–207. (doi:10.1016/j.ydbio.2017.02.017)
Nagano M, Avarbock MR & Brinster RL 1999 Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biology of Reproduction 60 1429–1436. (doi:10.1095/biolreprod60.6.1429)
Nakagawa T, Nabeshima Y-I & Yoshida S 2007 Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Developmental Cell 12 195–206. (doi:10.1016/j.devcel.2007.01.002)
Naughton CK, Jain S, Strickland AM, Gupta A & Milbrandt J 2006 Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biology of Reproduction 74 314–321. (doi:10.1095/biolreprod.105.047365)
Oakberg EF 1971 Spermatogonial stem-cell renewal in mouse. Anatomical Record 169 515–531. (doi:10.1002/ar.1091690305)
Okada Y, Scott G, Ray MK, Mishina Y & Zhang Y 2007 Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis. Nature 450 119–123. (doi:10.1038/nature06236)
Qu Y, Lu DY, Jiang H, Chi XC & Zhang HQ 2016 EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI. Nucleic Acids Research 44 7659–7672. (doi:10.1093/nar/gkw463)
Roy A, Lin YN, Agno JE, DeMayo FJ & Matzuk MM 2007 Absence of tektin 4 causes asthenozoospermia and subfertility in male mice. Faseb Journal 21 1013–1025. (doi:10.1096/fj.06-7035com)
Schonhoff SE, Giel-Moloney M & Leiter AB 2004 Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types. Developmental Biology 270 443–454. (doi:10.1016/j.ydbio.2004.03.013)
Shen X, Liu Y, Hsu Y-J, Fujiwara Y, Kim J, Mao X, Yuan G-C & Orkin SH 2008 EZH1 Mediates Methylation on Histone H3 Lysine 27 and Complements EZH2 in Maintaining Stem Cell Identity and Executing Pluripotency. Molecular Cell 32 491–502. (doi:10.1016/j.molcel.2008.10.016)
Song H-W & Wilkinson MF 2014 Transcriptional control of spermatogonial maintenance and differentiation. Seminars in Cell and Developmental Biology 30 14–26. (doi:10.1016/j.semcdb.2014.02.005)
Sun F, Xu Q, Zhao D & Chen CD 2015 Id4 marks spermatogonial stem cells in the mouse testis. Scientific Reports 5. (doi:10.1038/srep16549)
Tachibana M, Nozaki M, Takeda N & Shinkai Y 2007 Functional dynamics of H3K9 methylation during meiotic prophase progression. EMBO Journal 26 3346–3359. (doi:10.1038/sj.emboj.7601767)
Wang YJ, Xiang W, Wang M, Huang T, Xiao XY, Wang L, Tao D, Dong LY, Zeng FQ & Jiang GS 2014 Methyl jasmonate sensitizes human bladder cancer cells to gambogic acid-induced apoptosis through down-regulation of EZH2 expression by miR-101. British Journal of Pharmacology 171 618–635. (doi:10.1111/bph.12501)
Yang YA & Yu JD 2013 EZH2, an epigenetic driver of prostate cancer. Protein and Cell 4 331–341. (doi:10.1007/s13238-013-2093-2)
Yin J, Leavenworth JW, Li Y, Luo Q, Xie H, Liu X, Huang S, Yan H, Fu Z & Zhang LY et al. 2015 Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity. PNAS 112 15988–15993. (doi:10.1073/pnas.1521740112)
Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, Yamamoto M, Suda T & Nabeshima Y 2004 Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Developmental Biology 269 447–458. (doi:10.1016/j.ydbio.2004.01.036)
Yoshida S, Nabeshima Y-I & Nakagawa T 2007 Stem cell heterogeneity – actual and potential stem cell compartments in mouse spermatogenesis. Testicular Chromosome Structure and Gene Expression 1120 47–58. (doi:10.1196/annals.1411.003)
Yoshinaga K, Nishikawa S, Ogawa M, Hayashi SI, Kunisada T, Fujimoto T & Nishikawa SI 1991 Role of C-kit in mouse spermatogenesis – identification of spermatogonia as a specific site of c-kit expression and function. Development 113 689–699.
Yu WF, Zhang GZ, Lu BC, Li J, Wu ZY, Ma HM, Wang HM & Lian R 2016 miR-340 impedes the progression of laryngeal squamous cell carcinoma by targeting EZH2. Gene 577 193–201. (doi:10.1016/j.gene.2015.11.045)
Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B & Hoog C 2000 The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Molecular Cell 5 73–83. (doi:10.1016/S1097-2765(00)80404-9)
Zhang RJ, Wang RJ, Chang H, Wu F, Liu CT, Deng DF & Fan WH 2012a Downregulation of Ezh2 expression by RNA interference induces cell cycle arrest in the G0/G1 phase and apoptosis in U87 human glioma cells. Oncology Reports 28 2278–2284. (doi:10.3892/or.2012.2033)
Zhang Y, Wang S, Wang XX, Liao SY, Wu YJ & Han CS 2012b Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells. Cell Research 22 773–776. (doi:10.1038/cr.2012.17)
Zhang J, Ji F, Liu YL, Lei XP, Li H, Ji GJ, Yuan ZQ & Jiao JW 2014 Ezh2 regulates adult hippocampal neurogenesis and memory. Journal of Neuroscience 34 5184–5199. (doi:10.1523/JNEUROSCI.4129-13.2014)
Zhang J, Taylor RJ, La Torre A, Wilken MS, Cox KE, Reh TA & Vetter ML 2015 Ezh2 maintains retinal progenitor proliferation, transcriptional integrity, and the timing of late differentiation. Developmental Biology 403 128–138. (doi:10.1016/j.ydbio.2015.05.010)
Zheng K, Wu X, Kaestner KH & Wang PJ 2009 The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Developmental Biology 9 11. (doi:10.1186/1471-213X-9-11)
Zhou Q, Nie R, Li Y, Friel P, Mitchell D, Hess RA, Small C & Griswold MD 2008 Expression of stimulated by retinoic acid gene 8 (Stra8) in spermatogenic cells induced by retinoic acid: an in vivo study in vitamin A-sufficient postnatal murine testes. Biology of Reproduction 79 35–42. (doi:10.1095/biolreprod.107.066795)
Zhu W-S, Tang C-M, Xiao Z, Zhu J-N, Lin Q-X, Fu Y-H, Hu Z-Q, Zhang Z, Yang M & Zheng X-L et al. 2016 Targeting EZH1 and EZH2 contributes to the suppression of fibrosis-associated genes by miR-214-3p in cardiac myofibroblasts. Oncotarget. (doi:10.18632/oncotarget.13048)
