Spermatogenesis is a complex cellular differentiation process including mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis. Sertoli cells, which locate within the seminiferous tubules, directly interact with germ cells and supply essential factors for developing spermatogenic cells [1
]. Sertoli cells secrete niche factors to promote the maintenance of spermatogonial stem cells [2
], produce regulatory factors to control meiosis [3
], and provide structural and nutritional supports to direct spermatid development [4
]. Defects in Sertoli cell function often cause abnormal spermatogenesis and sterility [5
]. From cell ablation studies, it has been recognized that the number of Sertoli cells determines testis size and daily sperm production [6
]. Sertoli cells also influence testicular blood vessel architecture and secretion of testosterone and estrogen [7
]. Therefore, revealing new insights into Sertoli cell biology is crucial for understanding animal spermatogenesis.
Sertoli cells are specified from a bipotential somatic precursor cells in early fetal stage through a Sry
(Y-linked testis-determining gene) and Sox9
(Sry-box containing gene 9) dependent genetic program [10
]. After specification, Sertoli cells expand in number rapidly during the fetal and early postnatal periods before gradually enter a terminal differentiated state after puberty [12
]. Thyroid hormone is the master regulator of Sertoli cell proliferation and maturation in rodents. Neonatal hypothyroidism extend murine Sertoli cell proliferation and a significant increase in Sertoli cell number and sperm production [14
]. Thyroid hormone has conserved functions because it also inhibits the mitosis of Sertoli cells in bull [15
], pig [16
] and other animal species [17
]. Follicle stimulating hormone (FSH) and activins stimulate Sertoli cell proliferation [18
]. Bone morphogenetic protein 7 (BMP7), Interleukin-1, and Insulin growth factor 1 (IGF1) are potent mitogens for Sertoli cells in vitro and conditional deletion of IGF-1R in Sertoli cells caused defects in Sertoli cell proliferation and increased apoptosis [20
]. These hormones and growth factors likely work with cell cycle inhibitors p27kip1, p21Cip1 and Rb1 in Sertoli cells. In the testis of p27 or p21 knockout mice, Sertoli cell number and daily sperm production were significantly increased [23
]. Deletion of retinoblastoma protein (Rb1) induced mature Sertoli cells to continue cycling, therefore, caused severe defects in spermatogenesis [24
]. Key cell cycle regulators that control Sertoli cell mitosis have been partially elucidated, however, transcription factors that direct Sertoli cell growth and maturation remain largely unknown.
Several transcription factors have been demonstrated to be essential for Sertoli cell proliferation. The major function of Rb1 is to suppress E2F transcription factors and knockout transcription factor E2F3 in Sertoli cells rescued the phenotype in Rb1 conditional knockout animals [25
]. Transcription factors upstream stimulatory factor (USF) 1 and USF2 are expression in Sertoli cells and Usf1
knockout mice showed defects in spermatogenesis [26
]. Zinc finger transcription factor kruppel-like factor (Klf) 4 is responsive to FSH stimulation and involved in Sertoli cell maturation and proliferation [27
]. Estrogen receptors ESR1 and ESR2 activate CCND1 to modulate Sertoli cell proliferation [28
]. Hyopoxia indicule factors (HIFs) are regulated by FSH and likely play roles in Sertoli cell proliferation [29
]. Among these transcription regulators, Rb1-E2F3 system is the decisive factor determining Sertoli cell proliferation [25
], therefore, identifying and elucidating functional roles of factors in the Rb1-E2f regulatory network may help expand the list of transcription factors in the regulation of Sertoli cell function.
Transcription factor E4F1, originally identified as a regulator of the viral E4 and E1A promoters [30
], interacts with Rb1 and plays crucial roles in cell proliferation and stem cell fate decisions [32
deficient embryos die at the peri-implantation stage due to defects in mitotic progression, chromosomal segregation and apoptosis [33
]. In quiescent cells, E4F1 binds to hypophosphorylated Rb1 to maintain cell cycle arrest [36
]. In line with this observation, overexpression of E4F1 in fibroblasts suppresses the progression from G1 to S phase [37
]. E4F1 controls cyclin A expression by repressing its promoter activity [34
]. In hematopoietic stem and progenitor cells, E4F1 directly interacts with the checkpoint kinase 1 (CHK1) to regulate cell cycle progression and apoptosis [38
]. Recent studies suggest that in addition to its role in cell proliferation, E4F1is a potent regulator of pyruvate and lactate metabolism [39
]. Despite these important findings, the functional role of E4F1 in Sertoli cell has not been determined.
Because the proliferation of Sertoli cells is tightly controlled and E4F1 is a key regulator of cell cycle progression, we hypothesized that E4F1 served crucial roles in Sertoli cells. Using a conditional mouse model, the present study showed that E4F1 expression was enriched in Sertoli cells and loss of E4f1 in Sertoli cells led to reduced Sertoli cell number and testis size. Fertility of E4f1 conditional knockout animals was impaired and Sertoli cells lacking E4F1 activity exhibited reduced mitotic index. Together, these findings indicate that transcription factor E4F1 is expressed in murine Sertoli cells and crucial for Sertoli cell mitotic progression and fertility.
2. Materials and Methods
All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Welfare and Ethic Committee at the Northwest Institute, Chinese Academy of Sciences. Amh-Cre
(JAX Stock No. 007915) mice were mated with E4f1flox/flox
line (Dr. Guy Sauvageau laboratory, University of Montreal, Montreal, Canada) [38
] to generate Amh-Cre; E4f1flox/+
male mice. Amh-Cre; E4f1flox/+
male mice were mated with E4f1flox/flox
to generate Amh-Cre
(designated hereafter as E4f1
cKO) male mice. Amh-Cre
littermates were used as controls. All mice were maintained on a mixed 129S2/SvPasCrl; FVB/N genetic background.
2.2. Fertility Test and Sperm Concentration Analysis
45 days old control or E4f1 cKO males were paired with adult wildtype female mice. One male was mated with three females for 3 months. Seven control and seven E4f1 cKO animals were used in the fertility test. Average litter size was recorded to assess fertility. For sperm count, epididymis was put in 1 mL in HEPES medium and shred to release sperm. Then we used hemocytometer to count sperm.
2.3. Histological Analysis
Testes were fixed in Bouin’s solution. After dehydration, tissues were embedded with paraffin (HistoCore Arcadia, Leica, Mannheim, Germany). Paraffin-embedded tissues were then cut for 5 µm by microtome (Leica RM2235, Leica, Mannheim, Germany). After rehydration, sections were stained with hematoxylin and eosin (H&E). Images were examined by using a microscope (ECLIPSE E200, Nikon, Tokyo, Japan), and captured by CCD (MS60, MshOt, Guangzhou, China).
2.4. Meiosis Analysis
Meiosis analysis was conducted as described previously [40
]. Briefly, the tunica albuginea was removed to release the seminiferous tubules into a hypotonic extraction buffer containing 30 mM Tris, 50 mM sucrose, 17 mM sodium citrate dehydrate, 5 mM EDTA, 0.5 mM DTT and 0.5 mM Pheylmethylsulfonyl fluoride (PMSF), pH 8.2–8.4 (pH set by using boric acid) for 20 min. Subsequently, a few seminiferous tubules were placed into 60 µL 100 mM sucrose and torn to pieces to release the cell using two 1 mL syringe needle. Then 20 µL cell suspension was overspread on adhesive slides that were dipped in 1% paraformaldehyde, pH 9.2(pH set by using boric acid), containing 0.15% Triton X-100 in ddH2
O. The slides were stored in a hot humid chamber overnight. The slides were then dried in the air after dipped in primary antibody dilution buffer solution (ADB) (1% bull serum albumin (BSA), 0.1% cold fish skin gelatin, 0.5% Triton X-100 in 0.01 M phosphate buffered saline (PBS)). 60 µL diluted antibody was placed onto the slides, sealed with glass coverslips, and put the slides into a humid chamber overnight at 37 °C. Next, we removed coverslips and immersed the slides into ADB for 1 h. The secondary antibody was diluted in ADB and placed 60 µL of the diluted antibody onto the slides. After sealed with a glass coverslip, the slides were put into a humid chamber overnight at 37 °C and washed in PBS for 1h. Finally, Hoechst33342 were added for 1 min and the slides mounted in 50% glycerol before examining under a microscope (Leica).
2.5. Immunohistochemistry (IHC) and Immunofluorescence (IF)
Testes were fixed in 4% paraformaldehyde (PFA) for immunohistochemistry and immunofluorescence. Sections were boiled in 10 mM sodium citrate (pH 6.0) for about 20 min for antigen retrieval and IHC for E4F1 expression was blocked with endogenous peroxidase with 3% H2
. PBS washed sections for 5 min three times. The sections were incubated in the 10% blocking serum for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Normal IgG was used for negative controls. After washed in PBS for three times (10 min each), the sections were incubated with secondary antibodies for 1 h at room temperature. The E4F1 expression was visualized with 3,3′-diaminobenzidine tetrachloride (DAB) solution. For detection of immunofluorescent signal, slides were added Hoechst33342 for 1 min, then wash in PBS. Digital images were captured with a microscope (Leica, Mannheim, Germany). Primary antibodies were listed in Supplementary Table S1
2.6. TUNEL Labeling
To determine the number of apoptotic cells, the sections of testis were processed for terminal deoxynucleotidy1 transferase-mediated dUPT nick end labeling (TUNEL) using a TUNEL staining kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Co-Immunofluorescent staining of SOX9 and TUNEL was performed using In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany). After the sections were incubated in the 10% blocking serum for 1h at room temperature, SOX9 antibody was added to labeling mixture of In Situ Cell Death Detection Kit, incubated 1 h at 37 °C, and washed in PBS 10 min for three times, and then incubated with 555-conjugated donkey anti-rabbit IgG for 1 h at room temperature. Hoechst33342 incubated the sections for 1 min. Digital images were captured with a microscope (Leica, Mannheim, Germany).
2.7. 5-ethynyl-2′-deoxyuridine (EdU) Assay
EdU (RIBOBIO, Guangzhou, China) treated mice at a dosage of 50 mg/kg body weight throught intraperitoneal injections. Two hours after EdU injections, testes were collected. Before EdU detection, immunofluorescent staining of TRA98 was carried out. Then EdU was detected according to the manufacturer’s instructions of Cell LightTM EdU Apollo 567 in vivo Kit (RIBOBIO, Guangzhou, China).
2.8. qRT-PCR Analysis
Total RNA was purified from mice testes by the Trizol method (Ambion, Austin, TX, USA). cDNA was synthesized using StarScript II First-strand cDNA Synthesis Mix With gDNA Remover Kit (GenStar, Guangzhou, China). The qRT-PCR analysis was performed on the ABI ViiA7 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green master mix (Genstar, Guangzhou, China), and Gapdh was used as an internal control. Primer sequences used for qRT-PCR assay are: E4f1 forward: CCAGATGAACCCATCACT; E4f1 reverse: TGCCCACTTCCAACAA; Gapdh forward: AGGTCGGTGTGAACGGATTTG; Gapdh reverse: TGTAGACCATGTAGTTGAGGTCA; p21 forward: GCAGATCCACAGCGATATCC; p21 reverse: CAACTGCTCACTGTCCACGG; Rb1 forward: CTTGAACCTGCTTGTCCTCTC; Rb1 reverse: GGCTGCTTGTGTCTCTGTATT
2.9. Statistical Analysis
Data are presented as means ± s.e.m. for more than three independent experiments and at least three animals were used for each genotype. Two hundred (200) spermatocytes were used to analyze process of meiosis and 100 seminiferous tubules were used for diameter measurement. The average number of tubules used to analyze Sertoli cell number per cord was 22 per mouse. The average number of Sertoli cells used to analyze germ cells number per Sertoli cell was 1000 per mouse. The number of cords used to analyze the percentage of EdU+ cells in SOX9+ cells was 20. The number of Sertoli cells used to quantify percentage of TUNEL+ Sertoli cells was 2000 for each genotype. And 3 sections were used for each animal. Differences between means were examined using the t-test function of GraphPad Prism 5 (GraphPad Software Inc., La Jolla, San Diego, CA, USA). Differences between means were considered significant at p < 0.05.
Sertoli cells serve central roles in supporting spermatogenesis and defects in Sertoli cell lineage specification and development cause problems in fertility in human and animals [1
]. Because Sertoli cells only proliferate during fetal and neonatal periods of development, the size of Sertoli cell pool is determined by early puberty [44
]. In this study, we showed that transcription factor E4F1 was enriched in murine Sertoli cells and played an important role in regulating Sertoli cell number and fertility.
Sertoli cells directly interact with germ cells and support different types of germ cells in the neonatal or adult testis. Sertoli cells participates all aspects of germ cell development in fetal and postnatal testes. Sertoli cells secret growth factors and cytokine to promote prospermatogonia to spermatogonia transition [45
]. Sertoli cells are major contributor of spermatogonial stem cell niche [46
]. In adult murine testis, spermatocytes and spermatids are quickly lost upon Sertoli cell removal [47
]. In the present study, we found that E4F1 deletion in Sertoli cells affected testis size, however, meiosis progression was normal and spermeiogenesis was not affected. We concluded that reduced fertility in E4f1
cKO animals was due to decreased sperm production, which is directly caused by a reduction in Sertoli cell population. This conclusion is further supported by the fact that average number of germ cells supported by one Sertoli cell was comparable between control and the conditional knockout animals. A similar phenotype is observed in FSH-deficient male mice. Fsh beta gene knockout males had smaller testis and reduced Sertoli cell number, however, they produce viable sperm and fertile [48
]. E4F1 expression is enriched in mature Sertoli cells but did not play a significant role in regulating the quality of spermatogenesis.
Sertoli cell proliferation is regulated by hormones, growth factors and cytokines during fetal and neonatal period of development. FSH stimulates Sertoli cell proliferation by activating cAMP/PKA/ERK1/2 and P13K/Akt/mTORC1 dependent-pathway [49
]. Relaxin increases the levels of proliferating cell nuclear antigen (PCNA) in Sertoli cell cultures by activating P13K/Akt and ERK1/2 pathway [50
]. And there is crosstalk between FSH and relaxin at the end of the proliferative stage in rat Sertoli cells [35
]. Transcription factor c-Myc may have a role in FSH-dependent regulatory network [51
]. FSH induces the expression of transcription factor klf4, however, knockout experiments revealed that Klf4 is dispensable for Sertoli cell proliferation [52
]. In cultured human smooth muscle cells, E4F1 expression is strongly induced by estrogen and it is recognized as an estrogen-responsive genes that control cell proliferation [53
]. It is unclear if a similar mechanism exists in testis, however, we can speculate that E4F1 works as a key transcription factor for FSH, estrogen and other factors involving Sertoli cell proliferation. The upstream factors and signaling pathways that induce E4F1 expression in Sertoli cells should examined in the future studies.
The action of E4F1 is likely independent of Rb1 in Sertoli cells. Rb1 Knockout in Sertoli cells causes severe defects in spermatogenesis and Rb1-decient Sertoli cells reenter cell cycle and undergo dedifferentiation [54
]. The phenotype caused by Rb1 inactivation in Sertoli cells can be rescued by E2F3 knockout [25
]. Transcription factor ARID4A is an Rb1 binding partner and together, these two proteins function to maintain BTB function [55
]. In the present study, deletion of E4F1 did not change BTB integrity. E4F1 regulates lactate metabolism in skeleton muscle cells [56
] and one of the major function of Sertoli cells is to supply lactate to developing germ cells [57
], we hypothesized that spermatids might be affected by E4f1
inactivation in Sertoli cells. However, developing of meiotic and postmeiotic germ cells appeared to be normal in the E4f1
conditional knockout animals. E4f1
inactivation in Sertoli cells may induce a redirection of the glycolytic flux towards lactate production and secretion, therefore did not affect spermiogenesis. From these data, we concluded that E4F1 is not required for Sertoli cell maturation and terminal differentiation.
Instead, E4F1 works as an important factor controlling Sertoli cell proliferation in neonatal testis. E4F1 interacts with CHK1 and cell cycle arrest caused by E4F1 deletion can be rescued by Chek1 overexpression [38
]. In preimplantation embryo, E4F1 promotes cell cycle progression and maintains genome integrity [33
]. Cancer cells lacking E4F1 is arrested in G2/M of cell cycle [58
]. In Sertoli cells, inactivation of E4F1 impaired G1-S transition and increased apoptosis, however, these cells were not completely arrested. These data suggest that E4F1 function is important but not essential for Sertoli cell mitosis and survival. Other transcription factors that determine Sertoli cells proliferation and function in neonatal testis remain to be identified.