Immature male (60–80 g), young female (3–4 months), and perimenopausal female (9–10 months) Sprague–Dawley rats were obtained from the Animal Center at Shanghai. All rats were kept under controlled temperature (30 ± 2°C) and light (14 h light, 10 h dark) conditions with rat chow and water ad libitum. All experiments were approved by the institutional animal committee of the Fuzhou General Hospital.
Isolation and identification of BMSCs
The femurs and tibiae were removed from immature male Sprague–Dawley rats. The bone marrow plugs were flushed with phosphate-buffered saline (PBS), layered over a Percoll solution (density 1.083), and separated by centrifugation at 2500 RPM for 20 min at room temperature. Mononuclear cells at the interface were recovered and washed twice with PBS. The cells were cultured with complete medium, consisting of DMEM/F12 (1:1) medium supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% CO2. At confluence, the cells were harvested for passage with 0.25% trypsin containing 0.02% EDTA. All experiments were performed using cells from the third to fifth passage.
BMSC surface expression of CD29, CD34, CD45, and CD90 was analyzed by flow cytometry. Briefly, cells were incubated with the R-phycoerythrin (PE)-labeled antibodies (BD Biosciences, San Jose, California, USA) and their corresponding isotype controls: PE-CD34 (no dilution), PE-CD29 (1:16 dilution), PE-CD45 (1:8 dilution), and PE-CD90 (1:8 dilution). After a 30-min incubation in the dark, the cells were isolated by centrifugation (800 g for 5 min) after which the supernatant was removed. After the cells were washed three times with 2 mL PBS, the cell suspensions were filtered with 200 μm mesh filters to make single cell suspensions. 1× 106Cells were next analyzed for surface marker expression by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). Samples in which the primary antibody was omitted were used as the gate for the forward scatter (FSC) versus side scatter (SSC).
BMSCs were seeded in 6-well plates at a density of 1 × 105 cells/well. After 24 h, the supernatant in one of the plates was replaced with control DMEM-HG medium supplemented with 10% FBS, 10-6 M dexamethasone, 0.5 mM 3-methyl-1-methyl xanthine, 10 μg/mL insulin, and 0.2 mM indomethacin. In the remaining plates, the supernatant was replaced with induction medium comprised of DMEM-HG medium supplemented with 10% FBS, 10-7 M dexamethasone, 10 mM beta-glycerophosphate, and 50 mg/mL vitamin C. The medium was replaced every three days. The cells cultured in control medium were stained with Oil red after two weeks; the cells cultured in induction medium were identified by Von Kossa staining after three weeks.
To evaluate the secretion of growth factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1), by cultured BMSCs, cells were seeded in 6-well plates at a density of 1 × 106 cells/well. After 24 h, the supernatant was replaced with serum-free medium consisting of DMEM/F12 (1:1) medium, 100 U/mL penicillin, and 100 U/mL streptomycin. After three days, the supernatant was collected, and cytokine concentrations were determined using enzyme linked immunosorbent analysis (ELISA) using kits purchased from Uscn (Wu Han, Hubei, China).
Isolation and identification of GCs
Immature female rats (3–4 weeks) were injected once subcutaneously with 60 IU of pregnant mare serum gonadotropin (Animal Drugs Factory, Huangzhou, Zhejiang, China) to induce the development of multiple antral follicles. After 48 h, the rats were sacrificed, and the ovaries removed. The GCs were isolated by needle puncture under an inverted microscope. After obtaining single cell suspensions, they were washed twice with PBS and cultured with complete medium, including DMEM/F12 (1:1) medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% CO2. Cell of the third passage were seeded on microscope slides for immunocytochemistry analysis to confirm their identity. Briefly, single GC suspensions (1 × 105 cells/mL) were fixed with 4% paraformaldehyde and washed three times with PBS (5 min/wash) and then incubated with 0.5% Triton X-100 for 20 min at room temperature. After washing three times with PBS (5 min/wash), cells were incubated with 3% H2O2 for 5 min at room temperature to eliminate endogenous catalase activity. Nonspecific binding was blocked by incubating the cells will normal goat serum diluted in PBS (1:50) for 20 min at room temperature. Next, samples were incubated with FSHR primary polyclonal antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS in a humidified chamber at 4°C overnight. Controls consisted of samples incubated in PBS alone. After washing three times with PBS (5 min/wash), secondary goat anti-rabbit lgG antibodies were added for 60 min at 37°C. After three washes with PBS (5 min/wash), samples were incubated with a horseradish peroxidase-labeled streptomycin solution for 30 min at 37°C prior to color development with the 3,3' Diaminobenzidine (DAB) chromogen. After the reaction was stopped by washing the samples with tap water, the samples were counterstained with hematoxylin for 20 s. Subsequently, the samples were washed, dehydrated, mounted with Permount (Fischer, Fair Lawn, NJ, USA), and analyzed using a Leica TCS-2 microscope (Leica Microsystems Ltd., North Point, Hong Kong, China) equipped with a D1X digital camera (Leica Microsystems Ltd., North Point, Hong Kong, China).
Effect of BMSCs on GC apoptosis in vitro
Prior to in vitro experiments, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Sigma-Alderich, St Lois, MO, USA) were used to identify the optimal concentrations of cisplatin as well as optimal treatment time without inducing toxicity. GCs (1 × 104 cells/well) in logarithmic growth were seeded onto a 96-well plate and incubated with various concentrations of cisplatin (0, 1.875, 3.25, 7.5, 15, and 30 mg/L). After 24, 48, 72, 96, or 120 h, 20 μL of MTT solution (5 mg/L) was added to each well for 4 h after which 150 μL DMSO was added into each well. The OD value of each well was determined at wavelength 490 nm using a Stat Fax 2100 Microplate Reader (Awareness Technology, INC., Palm City, FL, USA).
Cultured GCs were separated into three groups: an untreated control group (control), a cisplatin (5 mg/L) treatment group (cisplatin), and group co-cultured with BMSCs and treated with 5 mg/L cisplatin (BMSC). GCs were seeded onto 6-well plates at a density of 5 × 105 cells/well. BMSCs were seeded on 6-well Millicell cell culture inserts (Millipore, Billerica, MA, USA) at a density of 2.5 × 105 cells/well. After 24 h, the supernatant was removed. In the control group, the medium was changed to 3 mL serum-free medium (DMEM/F12 medium containing 100 U/mL penicillin and 100 U/mL streptomycin). In the cisplatin group, cells were incubated with 3 mL serum-free medium with cisplatin (5 mg/L). In the BMSC co-culture group, cells were co-cultured with BMSCs and incubated with 3 mL serum-free medium with cisplatin (5 mg/L). After 72 h, the GCs were harvested and analyzed for apoptosis using an annexin V/PI apoptosis detection kit (MultiSciences Biotech, China) according to manufacturer’s protocols and flow cytometry (Beckman Coulter Epics XL Flow Cytometer; GMI, Inc.; Ramsey, Minnesota, USA).
Real time PCR analysis
GCs from each of the three treatment groups were also harvested to extract RNA. Bcl-2, bax, survivin, c-myc, p21, and GAPDH (housekeeping gene) mRNA expression was examined by real-time PCR using the following primers: and GAPDH mRNA expression was serving as control. The primers sequences are listed as follows: GAPDH (180 bp) sense, 5’-AAGGT CATCCCAGAGCTGAA-3’ and antisense, 5’-CTCAGTGTAGCCCAGGATGC-3’; c-myc (166 bp)sense,5’-TCTCTTCTTCCTCGGACTCG-3’and antisense, 5’-GGTTGCCTCTTTT CCACAGA-3’; p21 (169 bp) sense, 5’-GAGTGCCTTGACGATACAGC-3’ and antisense, 5’-CATGTACTGGTCCCTCATTGC-3’; survivin (180 bp) sense, 5’-GGAGCATAGG AAGCACTCCCCTG-3’ and antisense, 5’-CTCCGGGTCTCCTCGAACTCTT-3’; Bcl-2 (173 bp) sense, 5’-AGTACCTGAACCGGCATCTG-3’ and antisense, 5’-CAGGTATG CACCCAGAGTGA-3’ and Bax (174 bp) sense, 5’-CTGCAGAGGATGATTGCTGA-3’ and antisense, 5’-GATCAGCTCGGGCACTTTAG-3’. PCR amplification conditions consisted of an initial denaturation step of 2 min at 93°C followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min using an ABI 7500 PCR machine (Applied Biosystems; Foster City, CA, USA). The CT values from real time PCR results were analyzed by the 2-△△CT method. The PCR products were validated by running in 120 V × 20 min electrophoresis conditions.
Effects of BMSCs on GC apoptosis in vivo
Rats reach sexual maturity at 6 to 8 weeks and perimenopause at 10 to 15 months. Female rats (9–10 months) were randomly divided into the following three groups (n = 15 per group): control group, BMSC group, and estrogen group. In addition, 15 adult female rats (3–4 months) were included as the young group. Prior to initiation of the experiment, vaginal smears of the female rats were obtained at 8:30 am every morning to confirm the perimenopausal status of the older rats according to the estrous cycle. Upon confirmation, BMSCs (1–2 × 106 cells in 2 mL) were injected via vena caudalis of rats in the BMSC group; the control and young groups received a 2 mL saline injection. This treatment was repeated after one week. Rats in the estrogen group received 0.158 mg/kg/d nilestriol (Shang Hai New Hua Lian Pharmaceutical CO.LTD, China) for 8 consecutive days by gavage administration. Five rats from each group were randomly sacrificed one and three months after the second transplantation, and their ovaries were removed. The left ovary was fixed in formaldehyde and sectioned for analysis of GC apoptosis using a TUNEL apoptosis assay kit (Roche, Branford, CT). Tissue sections from each animal were examined by microscopy, and at least 100 GCs per field were counted in five randomly selected fields. The GC apoptosis rate was calculated as the number of apoptotic GCs divided by the total number of GCs. Cells were counted independently by two individuals who were blinded to the treatment group. The apoptotic index was defined as the average GC apoptosis rate from counted five randomly selected fields.
Quantitative data are presented as mean ± SD. Differences in gene expression among the three groups (control, cisplatin, and BMSC groups) were detected by one-way analysis of variance (ANOVA) with Tukey’s range test. The GC apoptosis rates were analyzed by two-way ANOVA to evaluate treatment effect (control, BMSC, estrogen) and age as well as time effect (1 month vs. 3 months) along with their interaction. The statistical analyses were performed with SAS software version 9.2 (SAS Institute Inc., Cary, NC). A two-tailed P-value < 0.05 indicated statistical significance.