Cell culture and transfection
GC-1 cells, a mouse-derived spermatogonia line, were purchased from ATCC (ATCC number: CRL-2053) and cultured in fresh Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum in 5% CO2 incubators at 37°C. Adherent cells were passaged every 2–3 days with 0.5 mg/ml trypsin (1:250) and 0.53 mM EDTA. All transfections were performed using Lipofectamin 2000 (Invitrogen).
An RNAi targeting sequence against mrhbdd1 (mouse homolog of rhbdd1) designated 6# (sense primer: GATCCGAATCAGCCTGACTTCAAATTCAAGAGATTTGAAGTCAGGCTGATTCTTTTTTA; antisense primer: AGCTTAAAAAAGAATCAGCCTGACTTCAAATCTCTTGAATTTGAAGTCAGGCTGATTCG) was cloned into pRNAT-H1.1/Hygro.
Male BALB/c mice and Sprague-Dawley rats were obtained from the Laboratory Animal Centre of PUMC (Beijing, China). The Animal Ethics Committee of the National Research Institute for Family Planning Beijing approved the animal experimentation protocols and all animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals established by the Chinese Council on Animal Care. The mice and rats were anesthetized under ether. The following organs were excised: intestine, muscle, spleen, liver, lung, heart, kidney, brain, epididymis, stomach and testis. The tissues were immediately immersed in liquid nitrogen and stored at -80°C pending use. Trizol reagent (Invitrogen) was used to isolate total RNA following the manufacturer's instructions. A SuperScript™ First-Strand Synthesis System (Invitrogen) was used for RT-PCR following the manufacturer's instructions.
Thirty μg of protein was loaded on each lane of a 12% SDS-polyacrylamide gel, separated and then electrophoretically transferred on to a PVDF membrane (GE Healthcare). The membrane was blocked in 5% skim milk for 1 h at room temperature and then incubated with polyclonal rabbit anti-RHBDD1 (Sigma), anti-HA (Sigma), anti-GFP (Santa Cruz), anti-caspase 3 (Santa Cruz) or anti-β-actin (Santa Cruz) overnight at 4°C. The membrane was incubated with anti-rabbit or anti-mouse HRP-IgG (Santa Cruz) for 1 h at room temperature. Chemiluminescence was detected using an ECL blot detection system (Santa Cruz).
Induction of apoptosis in GC-1 cells
For UV-induced apoptosis, mRHBDD1 knockdown or negative control stable GC-1 cells were exposed to UV irradiation (65 mJ). The medium was changed and cells were harvested 4, 5, 6, 7, 8 or 12 h post UV treatment. For PS341-induced apoptosis, cells were treated with 1 μM PS341 and were harvested 12 h post treatment.
Cells were removed from the plates using 0.25% trypsin-EDTA at 37°C for 5 min, collected, and fixed with 75% ethanol for 30 min at 4°C. They were stained with 50 μg/ml propidium iodide and 100 μg/ml RNase A in PBS at 37°C for 20 min. The DNA content of 10,000 cells was analyzed using a COULTER flow cytometer (EPICS-XL) with EXPO32-ADC software. The percentage of apoptotic cells (% of total cells) was analysed by the program EXPO32-ADC and shown as a bar chart (Microsoft Excel).
Cell proliferation assay
Cells were seeded at a density of 1000/well in 96-well plates in DMEM with 10% FBS. The medium was changed regularly, and measurements of proliferating cells were performed once per day for 5 days. For the MTS assay (Promega), 20 μl of a combined MTS/PMS solution was added to each well containing 100 μl culture medium and incubated for 3 h at 37°C, then the absorbance was measured at 490 nm using an ELISA plate reader. For the CCK-8 assay (Dojindo Molecular Technologies), 10 μl of CCK-8 solution was added to each well containing 100 μl culture medium and incubated for 3 h at 37°C, then the absorbance was measured at 450 nm using an ELISA plate reader.
Regeneration of spermatogenesis by GC-1 stable transfectant cells in mouse seminiferous tubules
BALB/c male mice were obtained from the Laboratory Animal Centre of PUMC (Beijing, China). The Animal Ethics Committee of National Research Institute for Family Planning Beijing approved the animal experimentation protocols and all animal experiments were performed according to guidelines (Guidelines for the Care and Use of Laboratory Animals) established by the Chinese Council on Animal Care. The mice were anesthetized under ether. Animals were maintained at 24 ± 1 °C, 55 ± 1% humidity with a 14 h Light: 10 h Dark cycle. Food and water were provided ad libitum. Male mice aged 6–7 weeks were treated with Busulfan (Sigma), 40 mg/kg i.p., to destroy the endogenous germ cells at 4–5 weeks prior to the transplantation. A recipient mouse was anesthetized and the testis was exteriorized through a midline abdominal incision. A sample of approximately 10 μL of cell suspension was injected into the recipient mouse testis by the rete testis, whereby approximately 60%–90% of the surface tubules were filled with cells. For each recipient mouse, one testis was used as the test site for the transplanted stably expressed pRNAT/H1.1/Hygro-negative and the contralateral testis was used for transplantation of the stably expressed pRNAT-H1.1/Hygro-6#.
At 8 weeks after transplantation, the treated and control testes were excised for histological examination. The testis tissues were embedded in O.C.T compound, snap-frozen in liquid nitrogen and cryosectioned. Forty sections were prepared from each testis and examined by fluorescent microscopy (Microphot Nikon) to identify the donor-derived germ cells. Sections were selected, fixed with 4% paraformaldehyde in PBS, stained with TRITC/Rhodamine conjugated peanut agglutinin (PNA) (Sigma) to visualize acrosomes, followed by staining with Hoechst 33258 (Sigma) to visualize nuclei and re-examined by confocal laser microscopy (Leica, Germany).
In our experiment, approximately 80% of the surface tubules were filled with cells in the control and test testis.
The results are expressed as means ± SD. Statistical comparisons used Student's t-test. P < 0.05 was considered statistically significant.