Comparison of Neonatal and Adult Mice-derived Sertoli Cells in Support of Expansion of Mouse Spermatogonial Stem Cells In vitro

All animal care and surgical interventions were undertaken in accordance with the approval of the Royan Institutional Review Board and Institutional Ethical Committee. Male mice (NMRI strain, Pasteur Institute, Tehran, Iran) of various ages (neonatal: 6 days; adult: 6-8 weeks) were used to isolate Sertoli cells. Six-day-old mice were used to isolate germ cells. 6-8week-old NMRI mice were used to make busulfan-induced infertile mice.

Isolation of TCs was performed as previously described (29) with slight modification. Briefly, the bilateral testes of 10-15 neonatal mice were collected, placed on ice and then transferred to the laboratory within 15 minutes following decapsulation. Seminiferous tubules were placed in a plate that contained Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) and separated from one another by pipetting for 5 minutes. The separated tubules were collected by centrifugation at 30×g for 2 minutes, then suspended in DMEM medium which contained enzyme solution that included 1 mg/ml of collagenase/dispase/hyaluronidase and 0.05 mg/ml DNase, for 10-15 minutes (with pipetting) at 37°C. All enzymes were purchased from Sigma-Aldrich. After a second centrifugation, in which the majority of interstitial cells were removed; a second digestion step was performed in DMEM by the addition of fresh enzymes to the seminiferous cord fragments. After filtration through a 40 μm nylon filter, isolated cells were maintained at 32ºC in an atmosphere of 5% CO2 in air for 10 days. The testis cells were cultured in DMEM medium supplemented with 13.5 g/l NaHCO3 (Sigma-Aldrich, USA), single-strength non essential amino acids, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 40 μg/ml gentamycin (all from Invitrogen, USA) with 1% fetal bovine serum (FBS) in the presence of 40 ng/ml of glial-derived neurotrophic factor (GDNF), 20 ng/ml mouse epidermal growth factor (EGF), and 10 ng/ml of human basic fibroblast growth factor (bFGF) (19).The culture medium was changed every three days.

Dissociated testis cells from testes of neonatal mice were allocated to six-well culture plates (1,000,000 cells/9 cm²). TCs derived from neonatal and adult mice were isolated by two-step enzymatic digestion with 1 mg/ml of collagenase (30). TCs in DMEM with the above supplements and 10% FBS were placed on datura stramonium agglutinin (DSA, 5 μg/ml, Sigma-Aldrich)-coated dishes and incubated for 1 hour at 37°C in a humidified atmosphere of 5% CO2 in air. After incubation, non-adherent cells were discarded and adherent cells fed by the culture medium for 5-7 days. After 5-10 days, the Sertoli cells formed a confluent layer. STO cells were used as a common feeder layer for culture of SSCs (1, 8, 22, 31). STO cells were cultured in DMEM with the above supplements and 15% FBS for 2-3 days after which their mitotic activation was halted with 10 μg/ml mitomycin.

Germ cells were isolated from the testes of six-day-old neonatal mice and cultured in vitro for ten days, then transferred to Sertoli cells isolated by DSA lectin from neonatal and adult mice and STO feeders for an additional five days. All cells were analyzed as following before and after co-culture.

Analysis of spermatogonial colonies was undertaken in two steps: the first step determined formed colonies ten days after primary culture, and the second step was accomplished five days after transferring the colonies onto three different feeder layers in order to compare the effects of feeder cells on the colonies’ properties.

The number of colonies in each six-well plate was counted by invert microscopy. Surface area of the colonies was measured with Image J software (National Institutes of Health); the number of cells per colony was measured with 100-150 colonies per group.

To evaluate cloning efficiency [(number of colonies/number of seeded cells) × 100], we analyzed the number of colonies of dissociated single spermatogonial cells. The colonies were mechanically separated from culture plates ten days after primary culture of TCs and singled by an enzyme solution that included trypsin-EDTA (0.05%; Invitrogen) and collagenase IV (1 mg/ml). Subsequently, 200,000 cells per group per replicate were seeded and colony efficiency was evaluated during a ten day period.

Total RNA was isolated using RNXtm (Cinagene, Tehran, Iran) and treated with a DNaseI, RNase-Free Kit (Fermentas) to remove genomic DNA contamination. One μg of total RNA was used for reverse transcription reaction with the Superscript II Reverse Transcriptase (Invitrogen) and random hexamer primer, according to the manufacturer’s instructions.

The primers used in this study were F: 5' CTT ATC CAA GTT CAC CAG TTC 3', R: 5' TGT ATA AGC CGG AGG TAT 3' for Dazl; F: 5' ACT CCA TTA AAC CAG GAA CCA 3', R: 5' CCC ATT TAA TCT CCT CCT TCT C 3' for Stra-8; and F: 5' GAT AAT CAT TTA GCA CAG CCT C 3', R: 5' GTC AAC AGA TGC AAA CAC AG 3' for mvh (vasa). As a positive control, Gapdh was used.

Single cells were obtained by trypsin from picked up SSC colonies on three different feeder layers and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 minutes. Single cells were rinsed with washing buffer (2% FBS in PBS plus 0.029 g EDTA) for 5 minutes prior to blocking in 10% normal goat serum in PBS for 15 minutes, followed by incubation with antibody solution overnight at 4°C. Primary antibodies were rat polyclonal anti-α6-integrin (1:100; Sigma), rat polyclonal anti-β1-integrin (1:100; Sigma-Aldrich) and mouse polyclonal anti-c-kit (1:200; Santa Cruz, CA). The following day, cells were washed twice with washing buffer for 5 minutes and incubated with the appropriate secondary antibody [goat anti-rat and goat anti-mouse labeled with fluorescein isothiocyanate (FITC; 1:200; Sigma-Aldrich)] for 45 minutes, and finally washed twice for 5 minutes before analysis. All steps were performed on ice.

Spermatogonial colonies that were cultured on three different feeder layers and confluent Sertoli cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH=7.4) for 20 minutes. Cells were washed twice with 0.1% Tween 20 in PBS prior to blocking in 10% normal goat serum (Vector, Burlingame, CA) in PBS for 15 minutes, followed by incubation with antibody solution against α6 and β1 integrins overnight at 4°C. The following day, cells were washed twice with 0.1% Tween-20 in PBS for 5 minutes, incubated with the appropriate secondary antibody for two hours, and subsequently washed with 0.1% Tween 20 in PBS for 5 minutes. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) in PBS.

To prepare recipient infertile mice testes, busulfan was administered by intraperitoneal injection to mice greater than 4 weeks of age, at a dose of 40 mg/kg, which would almost completely abolish spermatogenesis in most mouse strains (32).

Cells for transplantation were obtained from cultured SSC colonies ten days after primary culture and from cultured SSC colonies five days after transfer onto NSCs. Cells were incorporated for 24 hours with BrdU (0.1 mM; Sigma-Aldrich) in culture medium to trace the transplanted cells. Colonies were picked up manually, singled by trypsin and counted. About 100,000 cells per 10 μl medium (plus 5 μl trypan blue to visualize the injection) were used to transplant into each testis.

The abdomens of the recipient mice were opened with a 1.5 cm midline incision. Male mice were placed on the platform and the left testis was exposed. After fixation of the testis, the fat pad of the testes was separated and the position of the rete-testis located. The pipette was inserted into the rete-testis and cells were injected into the tubules. Tubular filling was monitored by observing the movement of the cell suspension which was facilitated by the addition of a small amount of trypan blue to the injection medium. Finally, the skin was sutured.

Recipient males were maintained for 4 to 5 weeks before analysis. Testes were removed and dissected from fat for analysis. The transplanted testes of the recipient mice were fixed in Bouin’s solution for 3-4 days, dehydrated and then embedded in paraffin. The sections were immunostained with a primary anti-BrdU (Sigma-Aldrich) such that donor cell-derived spermatogenesis could be visualized.

The results were expressed as mean±SD. All statistical analyses were conducted using SPSS (version 16) software (SPSS, Inc., Chicago, IL; http://www.spss.com). The statistical significance between the mean values was determined by one-way ANOVA and Duncan’s post-test. P ≤ 0.05 was considered significant.