PTEN signaling is required for the maintenance of spermatogonial stem cells in mouse, by regulating the expressions of PLZF and UTF1
- Wei Zhou†1, 2,
- Hongfang Shao†3,
- Di Zhang†2, 4,
- Jian Dong2,
- Wei Cheng5,
- Lu Wang2,
- Yincheng Teng3Email author and
- Zhuo Yu2Email author
© Zhou et al. 2015
Received: 21 April 2015
Accepted: 11 July 2015
Published: 28 July 2015
Pten plays a crucial role in the stem cell maintenance in a few organs. Pten defect also causes the premature oocytes and ovary aging. We and other groups have found that the phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling regulates the proliferation and differentiation of spermatogonial stem cells (SSCs). PTEN functions as a negative regulator of the PI3K pathway. Thus, we thought that the fate of SSCs might be controlled by Pten.
We report that promyelocytic leukaemia zinc finger (PLZF) and undifferentiated embryonic cell transcription factor 1 (UTF1), both of which are germ cell-specific transcriptional factors, are regulated by Pten. Conditional deletion of Pten leads to reduction in PLZF expression but induction of UTF1, which is associated with SSCs depletion and infertility in males with age.
Our data demonstrate that Pten is required for the long-term maintenance of SSCs and precise regulation of spermatogenesis in mouse. The finding of a Pten-regulated GFRα1+/PLZF−/UTF1+ progenitor population provides a new insight into the precise mechanisms controlling SSC fate.
KeywordsPten knockout PLZF UTF1 Spermatogonial stem cells PI3K-Akt signaling
Stem cells are capable of renewing themselves to maintain a stem cell pool as a preserved cell source for tissue homeostasis, while they can also differentiate into mature cells to carry out the function of a specific tissue. The precise balance of self-renewal and differentiation of stem cells is critical for the maintenance and function of a tissue or organ throughout life-time. Similar to other stem cells, spermatogonial stem cells (SSCs) renew themselves and meanwhile undergo a dramatic differentiation process-spermatogenesis to generate a large number of sperms consistently. Prior to spermatogenesis, multiple mitotic divisions of SSCs produce subpopulations of SSCs, and the balance of the SSC subpopulations is critical for long-term sperm production. Multiple proteins, such as promyelocytic leukaemia zinc finger (PLZF), GDNF family receptor alpha-1 (GFRα1) and undifferentiated embryonic cell transcription factor 1 (UTF1), are expressed in SSC subpopulations, which plays a crucial role in the maintenance of SSC pool. PLZF and GFRα1 are required in germ cells for stem cell self-renewal [1–3], whereas UTF1 is restricted to a small subset of spermatogonia that make the cells maintain the ability of differentiation [4, 5].
PTEN signaling is critical in governing the stem cell pool not only in the blood system and central neural system but also in reproductive system [6–8]. The loss of Pten in ovary via conditional knockout triggers premature of oocytes and ovary aging . On the other hand, we and other groups have revealed that the phosphatidylinositol-3-OH kinase(PI3K)/Akt/S6 pathway is a critical signaling in controlling the proliferation and division of SSCs. Disruption of this signaling or Akt knockout leads to the loss of spermatogonial cells and infertility in males [9, 10]. PTEN is a major negative regulator of PI3K signaling [11, 12]. To understand the function of Pten in regulating SSC fate and fertility in male mouse, we generated conditional Pten knockout males using germ cell specific Cre strain, the Stra8-Cre mouse. It was turned out that the loss of Pten caused reduction of PLZF expression, but induction of UTF1. Thus, conditional Pten knockout leads to depletion of SSC pool and infertility with age.
Conditional deletion of Pten in spermatogonial cells in mice
Conditional deletion of Pten caused overgrowth of testes followed by shrinking and sterility with age
Loss of Pten led to reduction of SSCs in neonatal males
The PTEN signaling regulated the expression of PLZF and UTF1 in SSCs
A putative model of SSC subpopulation fate controlled by Pten
Prior to undergoing differentiation of meiosis, spermatogonial stem cells proliferate and form a pool of cells at different division status to meet the dynamics of spermatogenesis. This cell pool is maintained by self-renewal and proliferation of SSCs and exists throughout life time. The transcriptional factor PLZF plays a crucial role in the maintenance of SSC pool in adult males, and PLZF knockout causes a progressive loss of spermatogonia with age [1, 2]. Interestingly, Pten knockout male pups have less PLZF positive SSCs even at 7 day-old (Figs. 4a, f). We have reported that PLZF expression is regulated by the PTEN signaling pathway in prostate cells . Similarly, PLZF expression was significantly reduced in the Pten knockout SSCs in this study (Fig. 5a), and further experiments in vitro confirmed that in SSCs, PLZF was indeed regulated by the PTEN signaling (Fig. 5b). Thus, in Pten −/− males, the loss of SSCs is partly due to the reduction of PLZF expression.
The precise regulation of the balance of self-renewal versus differentiation of stem cells is critical in controlling tissue homeostasis and function. Excessive differentiation-associated proliferation leads to depletion of stem cells and degeneration of tissue with age. In Pten −/− males, although the number of SSCs decreased, which occurred as early as 7 days after birth, testes underwent overgrowth or premature of larger size until day 42 but shrank afterward. This phenotype is apparently associated with excessive differentiation-proliferation of SSCs, which disturbs the long-term maintenance of stem cell pool, thereby leading to the exhaustion of spermatogenesis with age. Furthermore, Pten knockout induced the expression of UTF1, which is expressed in a subpopulation of spermatogonial cells in the testis [4, 16]. UTF1+ cells were significantly increased in the testis as early as at day 10 in the Pten −/− males compared with wild-type males (Fig. 4d). Using immunostaining of a nearly infertile 32 day-old Pten −/− testis, many UTF1+/PLZF- cells and few UTF1−/PLZF+ cells (Additional file 1: Figure S1A) were observed, indicating that Pten-deletion-induced UTF1 expression might boost SSCs differentiation associated with the testes overgrowth of Pten knockout males. Moreover, it has been reported that UTF1 makes the spermatogonia maintain the ability of differentiation  and is involved in the initiation of ES cell differentiation . All of these results indicate that UTF1+ cells are differentiating SSCs. To further identify the properties of UTF1+ cells, we performed a three color whole-mount staining of UTF1 with GFRα1 and PLZF of 7 day old tubules to locate UTF1 expression in the SSC population. Notably, a subpopulation of UTF1+/GFRα1+/PLZF− SSC and UTF1low/GFRα1+/PLZF+ cell was observed, which seemed to come from the same precursor cell through asymmetric division. Similar subpopulation cells in Pten −/− tubule lost both GFRα1 and PLZF expression. Therefore, we hypothesized a model of SSC fate in Fig. 6c. Thus, in this study, Pten knockout induced UTF1 expression in addition to causing the loss of PLZF expression. However, further studies should be conducted to address the mechanism how Pten regulates PLZF and UTF1 expression.
Although the testes overgrowth in Pten knockout males occurred within the first 2 months, the fertility and embryos production were lower compared with wild type males at same ages (Fig. 2b). Apparently, this phenotype is associated with the abnormality of sperms found in the epididymis which lack tails (Fig. 3b). This abnormality may be caused by the differentiation defects before haploid stages or during spermatogenesis because Pten is actively expressed in the haploid cells in the testis (Fig. 1d).
Collectively, The Pten-deletion-induced reduction of PLZF and increased expression of UTF1 apparently disturb the balance of self-renewal and differentiation of SSCs, leading to the depletion of spermatogonial cells and infertility with age.
By studying the model of Pten knockout in SSCs, we found that Pten is required for the long-term maintenance of SSCs and spermatogenesis. Our study provides a new insight into the precise mechanisms controlling SSC self-renewal versus differentiation to maintain SSC pool and spermatogenesis throughout life time, especially the discovery of a Pten-regulated GFRα1+/PLZF−/UTF1+ progenitor population might lead to a new understanding of SSC fate control.
Stra8-cre mice (Stock number 008208) and Pten LoxP/LoxP mice (Stock number 006440) were purchased from the Jackson Laboratory. Stra8-cre males were crossed with Pten f/f females to generate Pten knockout in SSCs. Genotype of Stra8-cre mice and Pten f/f mice were determined by PCR analysis using the primers and procedures provided by the Jackson Laboratory or by a previous research . For Pten PCR, the Pten f/f (1.1 kb) and Pten (1 kb) fragments were amplified by using the following primers: 5′-ACTCAAGGCAGGGATGAGC-3′ (forward), 5′-AATCTAGGGCCTCTTGTGCC-3′ (reverse). For Stra8-cre PCR, the Stra8-cre (179 bp) and Interleukin 2 (Il2 internal positive control, 324 bp) fragments were amplified by using primers: 5′-GTGCAAGCTGAACAACAGGA-3′ (Stra8-cre forward), 5′-AGGGACACAGCATTGGAGTC-3′ (Stra8-cre reverse); 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (Il2 forward), 5′-GTAGGTGGAAATTCTAGCATCATCC-3′ (Il2 reverse). Animals used in this study were maintained according to the Guide for the Care and Use of Laboratory Animals (Publication 85-23, revised 1996; National Institutes of Health, Bethesda, MD, USA), and the protocol was approved by Shanghai Jiao Tong University School of Medicine (Shanghai, China)
Histological analysis and immunostaining
Testes and epididymis were fixed in fresh Bouin’s fixative, embedded in paraffin and sectioned at 4 μm thickness. After the hematoxylin and eosin staining, the sections were mounted and viewed under a microscope (Carl Zeiss, Maple Grove, MN, USA).
For immunohistochemical staining, testes were fixed in 4% paraformaldehyde, embedded in OCT and sectioned at 8 μm thickness. The endogenous peroxidase activity was blocked by placing the slides in 3% hydrogen peroxidase for 10 min followed by a tap water rinse. After being blocked with 5% BSA, slides were subsequently incubated with the primary antibody against PTEN (1:50 dilution, BOSTER BA1377) at 4°C overnight, slides were then incubated with Biotin conjugated secondary antibody. Following incubation with Streptavidin-Biotin Complex (BOSTER SA1022), visualization was performed with a DAB reaction, thereby resulting in brown staining of structures containing the epitope. Cellular nuclei were counterstained with hematoxylin and slides were permanently mounted and evaluated under a light microscope. For immunofluorescent staining, after blocking with 2% BSA, frozen slides or cell slides were stained with antibodies against PLZF (1:100 dilution, R&D, AF2944), UTF1 (1:1,000 dilution, ABCAM, ab24273) or GFRα1 (1:40 dilution, R&D, AF560). The primary antibodies were revealed with Alexa-555 and Alexa-488 conjugated secondary antibodies together with DAPI to stain the nuclei. The sections were mounted and viewed under a fluorescence microscope. For statistical analysis, five different slides from Pten +/+ or Pten −/− mice were stained and positive cell numbers were calculated and analyzed by one-way ANOVA (α = 0.05). For whole-mount staining, with enzymatic dissociation of the testes using 1 mg/ml collagenase for 5 min at 37°C, untangled seminiferous tubules were fixed with 2% paraformaldehyde containing 0.5 mM CaCl2 for 30 min at room temperature. After incubation with 1% Ttriton 100 for 10 min, samples were dehydrated through a series of methanol (25, 50, 75, and 100% in PBS containing 0.5% Triton 100—PBS-T) on ice followed by rehydration in PBS-T. The seminiferous tubules were incubated in a blocking buffer (1% BSA and 4% donkey serum) for 1 h and incubated with the first antibody combination at 4°C overnight. The appropriate second antibodies (Alexa-555, Alexa-488 and Dylight-405 conjugated) were applied onto the samples at room temperature for 2 h. After washing with PBS-T, the samples were mounted and observed under a fluorescence microscope.
Western blot analysis
The proteins were extracted from the cells or testes using the lysis buffer containing 50 mM Tris-HCl (pH7.4), 1 mM EDTA, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 10 mM sodium fluoride, 1 mM sodium orthavanadate and 1% protease inhibitor cocktail (Sigma-Aldrich Corp, St. Louis, MO, USA). The extracted samples containing 50 μg proteins were subjected to 10%SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The filter was probed with PLZF antibody (1:200 dilution, R&D AF2944), UTF1 antibody (1:250 dilution, Chemicon MAB4337), PTEN antibody (1:1,000 dilution, Millipore 04-035), GFRα1 antibody (1:2,000 dilution, R&D AF560), P-AKT antibody (1:1,000 dilution, Cell Signaling Technology #4058s) and β-actin (Cell Signaling Technology). Appropriate secondary antibodies were used and the antibody-antigen complexes in the membranes were visualized using an enhanced-chemiluminescent detection kit (Millipore). The images were scanned using LAS-4000 mini (FUJIFILM, Minato-ku, Tokyo, Japan).
RNA isolation and RT-PCR analysis
The total RNAs were extracted using TRIzol reagent (Invitrogen) and then the RNAs were reverse transcribed by using a Reverse Transcription kit according to manufacturer’s instructions (TaKaRa, DRR037A). The following primers were used for SYBR Green–based real-time PCR (TaKaRa, DRR420A) on a 7900HT Real Time PCR System (Applied Biosystems Inc, USA): Gapdh [GenBank: NM_008084.3], 5′-TGCCCCCATGTTTGTGATG-3′ and 5′-TGTGGTCATGAGCCCTTCC-3′; Pten [GenBank: NM_008084.3], 5′-TTCATACCAGGACCAGAGGA-3′ and 5′-TTGTCATTATCTGCACGCTCT-3′. Relative gene expression was calculated by the two DDCt method against internal reference gene of glyceraldehyde-3-phosphate dehydrogenase (Gapdh).
In vivo fertility assay
To evaluate the effect of Pten −/− on fertility, we carried out in vivo fertility assay. For each experiment, two normal female mice were mated with one Pten +/+ or Pten −/− male for 2 weeks and then embryos were counted. This mating test was artificially divided into three groups according to the male’s ages as follows: 35–60 days, 61–100 days, and older than 100 days. All statistical analyses were conducted with GraphPAD 5.0.
Isolation of haploid cells
Testes were cut into pieces after removing the tunica albuginea, and testicular fragments in PBS were shocked roughly to wash out the intermediate cells near the lumen. Subsequently, the cells in supernatant were collected and stained with Hoechst 33342 (5 μg/ml). After 90 min of incubation, cells were resuspended in an ice-cold cell solution (PBS with 10% FBS) containing 2 μg/ml of propidium iodide for dead cell discrimination. All the solutions contain verapamil (50 μM/ml) to block the efflux of Hoechst. Finally, sorting was performed on an Influx cell sorter with UV laser (BD Biosciences) .
Isolation and culture of spermatogonial stem cells
Testes were removed from pups with fine forceps using sterile procedures and cut into pieces after removing the tunica albuginea. Following a two-step enzymatic digestion at 37°C until the tubules became minimum, supernatants were pipetted and collected quickly. The supernatant was centrifuged to remove the collagenase and the cells were incubated in a dish for 1 h, when the somatic cells had adhered to the bottom of the dish, the supernatants were collected and resuspended in KO-DMEM medium containing 1% FBS and 1,500 units/ml LIF to 6 well plates (for western blotting) or to 12 well plates with covers in each well (for immunofluorescent staining). Recombinant human GDNF and bFGF were added at a final concentration of 20 and 1 ng/ml respectively. Cells were maintained at 34°C in a humidified 5% CO2 atmosphere . The medium (containing 5 μM PI3K inhibitor or rapamycin 20 nM and growth factors) were changed every other day.
phosphatase and tensin homolog
promyelocytic leukaemia zinc finger
undifferentiated embryonic cell transcription factor 1
spermatogonial stem cells
stimulated by retinoic acid gene 8
enhanced green fluorescent protein
GDNF family receptor alpha-1
forkhead box O3
leukemia inhibitory factor
fetal bovine serum
glial cell-derived neurotrophic factor
basic fibroblast growth factor
DZ, JD helped with the mice manipulation, WC and LW did the SSC isolation and culture experiments, WZ performed most of the experiments, HS and YT prepared the manuscript, ZY and WZ designed the research and prepared the manuscript. All authors read and approved the final manuscript.
The other authors wish to dedicate this paper to Dr. Lixin Feng who passed away during the manuscript preparation. This study was supported by Grants from the National Natural Science Foundation of China (31171409, 81000049, 81370654), National Key Basic Research and Development Program of China (2012CB966603), Innovation Program of Shanghai Municipal Education Commission (13G20), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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