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.