Table 1.
PCR primer sequences.
Figure 1.
The characterization and differentiation of mouse SMSCs.
Morphology of A–B) F-SMSCs (a cell cluster in the middle of the dish) and C–D) M-SMSCs from RFP transgenic mice (scale bar, 200 µm). E) Phenotype of F-SMSCs by flow cytometry. Of the F-SMSCs, 96% expressed CD73 but not CD45, CD34, or CD14. F) M-SMSCs detected by flow cytometry. Of the M-SMSCs, 92% expressed CD73 but not CD45, CD34, or CD14. G) F-SMSCs and H) M-SMSCs were able to differentiate into adipocytes (oil red O staining), chondroblasts, and osteoblasts under standard in vitro differentiation conditions. The nuclei were counterstained with DAPI (blue) (scale bar, 50 µm).
Figure 2.
Changes in total body weight and weight of reproductive organs in treated, untreated control, and normal control animals over 8 weeks.
A) Representative photograph depicting the body type of 12 week-old mice in the (a) untreated control group, treated groups (b, F-SMSCs; c, M-SMSCs), and (d) normal control group. B) The total body weight of untreated mice decreased over the study period. However, mice in both the F-SMSC-treated and M-SMSC-treated groups weighed significantly more than mice in the untreated control group from the second week onward (**, P<0.01). C) Representative photograph of ovaries removed from (a) the untreated control group, treated groups (b, F-SMSCs; c, M-SMSCs), and (d) normal control group. D) As indicated, the total weight of ovaries in the treated groups (both M-SMSC- and F-SMSC-treated) showed remarkable increases over the study period except before the first week (**, P<0.01). E) The total weight of the uteruses in the treated group was significantly higher than that of untreated controls from the third week onward (*, P<0.05; **, P<0.01). F) There was no obvious change in the total weight of the cervixes and vaginas during the study period until after the 7th and 8th weeks (**, P<0.01).
Figure 3.
Offspring produced by mating after SMSC transplantation into the mice sterilized by chemotherapy and those produced by untreated controls and normal controls.
A) Litters of the different groups. B) Distribution of offspring in the treated and untreated groups. On average, significantly more pups were born to the mice that received chemotherapy plus F-SMSC- or M-SMSC-transplantation than to untreated controls (**, P<0.01). However, the average number of pups produced by the normal control mice was still higher than the average number of pups from mice receiving F-SMSC- or M-SMSC-transplantation (**, P<0.01).
Table 2.
Summary of female fertility study.
Figure 4.
Histological evaluations of ovaries in treated, untreated control, and normal control animals by midline section and HE staining.
Ovary sections from sterilized non-transplanted mice after (A) a 7-day recovery period and (B) a 14-day recovery period, which showing the hollow structure destroyed by chemotherapy. Ovary sections from sterilized non-transplanted mice showing fibrosis and reduced hollow structure after a (C) 28-day recovery period and (D) 2-month recovery period. Ovary sections from sterilized recipient mice at (E) 7 days, (F) 14 days, (G) 28 days, and (H) 2 months after transplantation of SMSCs. Primordial follicles are visible in F (arrows), developing follicles at various stages are visible in G and H. However, there were fewer such developing follicles in G than in H. The morphology of ovary section of normal (I), untreated (J), and treated (K) control animals. (H) The changes in the ovarian volumes in treated, untreated control, and normal control (**P<0.01, *P<0.05). Scale bars: (F) 100 µm, (A–E, G–H) 200 µm, (I–K) 500 µm.
Figure 5.
Transplantation of a line of RFP-transgenic SMSCs into chemotherapy-sterilized recipient mice.
A) Immunofluorescence of ovarian sections from sterilized mice 7 days after transplantation with RFP-transgenic SMSCs. Arrows indicate RFP staining in the ovarian stroma. B) RFP staining was observed around the antral follicle 21 days after transplantation. Asterisks indicate the antral follicle. C) RFP-positive cells were observed around granulosa cells in recipient ovaries 28 days after SMSC transplantation. D) RFP staining was observed in antral follicles of recipient ovaries 2 months after SMSC transplantation. Scale bars: A, C) 50 µm; B, D) 100 µm; A, insets) 20 µm; and B, D insets) 10 µm.
Figure 6.
Double-staining of RFP and AMH in antral follicles of recipient ovaries 2 months after SMSC transplantation.
A) DAPI. B) RFP staining cells. C) AMH staining cells. D) Merged image of A, B, and C. Scale bars: 50 µm; insets) 20 µm.
Figure 7.
SMSC administration decreased ovary inflammation and improved folliculogenesis transcription.
A) Analysis of pro- and anti-inflammatory cytokine gene expression as performed by qPCR. Administration of SMSCs 7 days after chemotherapy resulted in significantly less gene expression for TNF-α, TGF-β, IL-8, IL-6, IL-1β, and IFN-γ than in untreated control mice (*P<0.05, **P<0.01, ***P<0.01). B) Expression of oocyte-specific genes in treated and untreated recipient mouse ovaries. Quantitative real-time RT-PCR showed that the relative expression of Nobox, Nanos3, and Lhx 8 was significantly higher in the SMSC group compared than among untreated controls (**P<0.01). 18s rRNA served as an internal housekeeping gene. Data represent mean±SE of 3 independent experiments.