POF mice model
Six-week-old female Institute of Cancer Research (ICR) mice were purchased from Huayikang Biotechnology Co., Ltd. All animals have free access to food and water. Vaginal smears were used to monitor the estrus cycle. These animals were randomly divided into sham, POI, single transplantation hUC-MSC group and multiple transplantation hUC-MSC group (n=30 in each group). In order to establish a POI model, mice from the POI, single injection of hUC-MSCs and multiple injections of hUC-MSCs were intraperitoneally injected with a mixture of 120 mg/kg cyclophosphamide and 30 mg/kg busulfan. The sham group was injected with saline only. The day of receiving chemotherapy drugs was recorded as the first day. Vaginal smear test to observe the estrus cycle. On the 7th day after chemotherapy, mice in the single injection of hUC-MSCs and multiple injections of hUC-MSC were injected with 100 μl of 2×106 hUC-MSCs cell suspension into the tail vein . And on the 14th and 21st days after chemotherapy, mice in the hUC-MSCs group injected multiple times were injected with 100 μl of 2×106 hUC-MSCs cell suspension. In the fertility testing experiment, we used mice in the sham group, POI, single injection of hUC-MSCs and multiple injections of hUC-MSCs 14 days and 60 days after chemotherapy (n=5 in each group), and caged according to the ratio of male to female 2:1. All procedures have been approved by the Animal Protection and Use Committee of the National Family Planning Society of China. This study was conducted in strict accordance with the recommendations in the "Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health".
Isolation and culture of hUC-MSCs
Human umbilical cord samples were obtained from full-term babies delivered by caesarean section in Haidian Maternal and Child Health Hospital, and the umbilical cord was washed with PBS to remove contaminated blood. Remove the umbilical cord to remove the arteries and veins, cut them into small pieces of 0.5-1 mm3, and place them in the bottom of the tissue culture dish. Place the petri dish in a 37°C, 5% CO2 incubator. Cells are cultured in MEM-Alpha complete medium (10% fetal bovine serum, 1% diabody). When the cells reach about 90% confluence, they are digested with 0.25% trypsin, and the 1:2 separation and passage are continued. And make the cells grow to the logarithmic growth phase [15, 16].
Human MSC Analysis Kit (BD, NJ, USA) is used to identify MSC. Cells are resuspended in 1×107 cells/ml buffer, 100 µl of prepared cell suspension was added to 9 tubes and incubated with various fluorescent dye-conjugated specific antibodies (FITC Mouse Anti-Human CD90 (tube 1); PE Mouse Anti-Human CD44 (tube 2); PerCP-Cy™5.5 Mouse Anti-Human CD105 (tube 3); APC Mouse Anti-Human CD73 (tube 4); Nothing (tube 5); hMSC Positive Isotype Control Cocktail and PE hMSC Negative Isotype Control Cocktail (tube 6,8); hMSC Positive Cocktail and PE hMSC Negative Cocktail (tube 7,9) in the dark for 30 min at room temperature. The results were analyzed by flow cytometry, and the experimental results were verified three times.
To promote osteogenic differentiation, the cells were seeded into twelve-chamber-slides and cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Utah, USA) containing 10% fetal bovine serum (FBS). When the cells reached 70%–80% confluence, replaced osteogenic differentiation medium (HyClone, Utah, USA) and changed every 3 days . Two weeks later, the calcified extracellular matrix was stained with 2% Alizarin Red to confirm osteogenic differentiation. The experiment was repeated three times.
To induce adipogenic differentiation, cells were cultured in MSCs medium until reaching 90% confluence. Then, the cells were induced in adipogenic induction medium (MEM Alpha Modification (HyClone, Utah, USA) for 3 days. After 3 days, adipogenic induction mediumwas replaced by maintenance medium containing sorely of 10 µM insulin and MSCs medium and changed every 3 days. For the negative control, the cells were cultured in MSCs medium . After 1 week, formation of intracellular lipid droplets was stained by Oil Red O staining. The experiment was repeated three times.
ELISA detection of mice estradiol (E2) and follicle-stimulating hormone (FSH)
In order to ensure that each group of mice estrus were in the same period, mice were administered by intraperitoneal injection of Pregnant Mare Serum Gonadotropin (PMSG) 2 days before euthanasia. Blood samples were collected on the 14, 21, 28, and 60 days after the POF mold. The samples were incubated overnight at 4°C, then were centrifuged for 5 min at 4000 rpm. The resulting supernatant sera were collected. The levels of E2 and FSH were respectively measured by Mouse E2 ELISA Kit (Bio-Swamp, Wuhan, China) and Mouse FSH ELISA Kit (Bio-Swamp, Wuhan, China). The experiment was repeated three times.
Histologic staining and follicle counting
The ovaries were fixed overnight in 4% paraformaldehyde, embedded in paraffin by dehydration, and serial sectioning was adopted. The thickness of the serial sections was 5μm, and every ten sections were counted. 20 sections were taken and stained with hematoxylin and eosin (H&E). The ovarian primordial follicles, primary follicles, secondary follicles and mature follicles were counted under TE2000-u reverse phase microscope (Nikon, Tokyo, Japan). The classification and characteristics of all levels of follicles are determined as follows: primitive follicle is close to the white membrane, and the oocyte is surrounded by a single layer of flat granular cells; primary growth follicle is surrounded by one or more layers of cubic granular cells, and red staining appears between the two zona pellucida, the follicular membrane of connective tissue appears on the periphery of the follicle; follicular cavity appears in the secondary growth follicle, and the follicular membrane can be separated into the inner and outer membranes; mature follicle has obvious cumulus, large follicular cavity, and many capillaries between the cells; Atresia follicle follicle wall collapses, egg cell structure is not clear, or even disappear, zona pellucida shrinks.
The paraffin sections were deparaffinized, rehydrated and high pressured in a citrate buffer (pH 6.0) for 2min to retrieve antigenicity. Then, the samples were incubated with 3% hydrogen peroxide 10min to quench endogenous peroxidase activity. Sections were blocked non-specific antigen with 10% goat serum. After that, sections were incubated with Anti-Ki67 antibody (1:100; Abcam, Cambridge, UK) or mouse anti-human anti-Müllerian hormone (AMH, 1:30; AbD Serotec, Oxford, UK) in a humidified chamber overnight at 4°C. Negative control were conducted with 10% goat serum overnight at 4°C alone. After incubating with primary antibody, peroxidase-conjugated affinipure goat-anti-rabbit IgG (1:200; ZSBIO, Beijing, China) or peroxidase-conjugated affinipure goat-anti-mouse IgG (1:200; ZSBIO, Beijing, China) were added for 1 hour at room temperature. Then, colourated with 3, 3-diaminobenzidin (DAB; ZSBIO, China) at room temperature without light for 10min and counterstained with hematoxylin for 10s. After sealing slides, the samples were photographed with microscope (Nikon, Tokyo, Japan). The experiment was repeated three times.
Ovary samples were collected and fixed in 4% paraformaldehyde overnight at 4°C, then, dehydrated with 30% sucrose 1 week. After that, samples were fixed with the Tissue-Tek OCT Compound (Sakura Finetek Middle East, Dubai, United Arab Emirates) at -80°C and sliced in 7 um thick sections at -25°C. Slides were blocked with 10% goat serum for 1 hour at room temperature. Slides were then incubated with mouse anti-human nuclei monoclonal antibody (1:100; Millipore, USA) at 4°C overnight. 10% goat serum as the primary antibody were used as negative controls. Sections were probed with TRITC–labeled IgG (1:200; ZSBIO, Beijing, China) and counterstained with 4', 6-diamidino-2-phenylindol (DAPI). Fluorescence images were taken through a fluorescence microscope (DMI3000; Leica, Heidelberg, Germany). The experiment was repeated three times.
Microarray hybridization and data analysis
According to the manufacturer's instructions, the miRNAeasy Mini Kit (Qiagen GmbH, Hilden, Germany) was used to isolate total RNA from the ovaries of three mice in the control, POF and hUC-MSCS groups, respectively. The miRNA microarray including probe labeling, hybridization, hybridization image scanning and initial data analysis was performed by LC Sciences (LC Sciences, Houston, Texas, USA). Use LC-miRHumanMouseRat_11.0_080411 array. The mRNA chip uses the OneArray chip (Tecenet, Chengdu, China). Use cyclic local weighted regression (LOWESS) method for normalization. t test was performed between the control group and the POF group, the POF group and the hUC-MSCs group, and the hUC-MSCs group and the control group, and the statistical analysis of the microarray data has been performed.
Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)
The Trizol (Invitrogen, CA, USA) method is used to extract RNA from ovarian tissue. The RNA was extracted with reverse transcription kit (TakaRa Biotechnology, Shanghai, China) reverse transcribed cDNA for qRT-RNA detection. The experiment was repeated at least 3 times less, and P<0.05 was considered statistically significant. Primer sequence: mRNA-FSHR-Forward, 5'-GAGGTGCAAGCCCAGATTTA-3'; mRNA-FSHR-Reverse, 5'-GAGGGACAAGCACGTAACTATT-3'; mRNA-INHIBINα-Forward, 5'-TCTGAACCAGAGGAGGAAGAT-3'; mRNA-INHIBINα-Reverse, 5'-GGGATGGCCGGAATACATAAG-3'; mRNA-INHIBINβ-Forward, 5'-AAGAAAGAGGTGGATGGAGATG-3'; mRNA-INHIBINβ-Reverse, 5'-CAGCATGAGGAAAGGTCTATGT-3'; mRNA-GAPDH-Forward, 5'-GGTGAAGGTCGGTGTGAACG-3'; mRNA-GAPDH- Reverse, 5'-CTCGCTCCTGGAAGATGGTG-3'
To determine significance between two groups, the unpaired Student’s t test was used. One-way ANOVA was used to calculate the significant differences among the groups. Prism6.0 software was used to paint. P value less than 0.05 was considered to be significant.