Fig 1: Growth factors administration into mouse ovaries. (a) Cytokine secretion from mouse adipose-derived mesenchymal stem cells (A-MSCs) measured by enzyme immunoassay (EIA). Secretion of mouse vascular endothelial cell growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) were measured by enzyme-linked immunosorbent assay (ELISA). (b) Fluorescence cell sorting (FACS) analysis of mouse A-MSCs stained with cell surface antigen markers of MSCs. (c) Ratio of number of follicles and corpus lutea (CL) in A-MSC and growth factor-transplanted left ovary vs -non-transplanted right ovary. V: VEGF; H: HGF; I: IGF-1; 1: 10 × dose; and 10: 10 × doses. Significant differences between each character each character is represented by a, b, c, d, e and f (P<0.05).
Fig 2: Cytokine secretion from adipose-derived mesenchymal stem cells (A-MSCs), cytokine expression in A-MSC-transplanted ovaries and the localization of A-MSCs in ovaries after transplantation. (a) Cytokine secretion (vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF) and estradiol) from A-MSCs. (A) VEGF; (B) IGF-1; (C) HGF; and (D) estradiol. Each cytokine was measured by enzyme immunoassay (EIA) analysis. Significantly higher levels of VEGF, IGF-1 and HGF (*P<0.05) were secreted from A-MSCs (n=7), compared with fibroblasts (n=9). No significant difference in estradiol levels was observed. Adipose: A-MSCs; fibroblast: tail fibroblast cells. (b) Quantitative real-time polymerase chain reaction (qRT-PCR) on ovaries after A-MSC and tail fibroblast cell transplantation. (A) VEGF; (B) IGF-1; (C) HGF; and (D) StAR. Adipose: A-MSCs; fibroblast: tail fibroblast cells (*significantly different, P<0.05). (c) Immunohistochemistry (IHC) of cytokines (VEGF, IGF-1 and HGF) in ovaries after A-MSC transplantation. Top panels: sham operation; middle panels: cyclophosphamide (CTX) injected; bottom panels: CTX and A-MSC injected. BM: bone marrow-derived MSCs; adipose: A-MSCs. Scale bar=200 µm. (d) Localization of A-MSCs after transplantation into ovaries by fluorescent in situ hybridization (FISH). Male A-MSCs were injected into ovaries and detected by FISH using the Y-chromosome-specific probe. Red: X chromosome conjugated with Texas Red; green: Y chromosome conjugated with FITC. (A) AF: antral follicles, GC: granulosa cells; (B) CL: corpus luteum; (C) T: thecal cells. Cells containing XY karyotype, indicating male-derived ASCs, were observed only in thecal layers.
Fig 3: The Hgf–Pik3ca–Mtor signal pathway was inhibited after Pdcd5 knockout. (a–c) The Hgf mRNA and protein levels in different embryos and placentas at E11.5 were detected by RT-PCR (a), qRT-PCR (b) and ELISA (c). (d–f) Proteins from Pdcd5+/+ or Pdcd5–/– embryos at E11.5 were extracted and subjected to western blot analysis using the indicated antibodies
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