Fetal liver mesenchymal stem cells restore ovarian function in premature ovarian insufficiency by targeting MT1

Abstract

Background: With the development of regenerative medicine and tissue engineering technology, almost all stem cell therapy is efficacious for the treatment of premature ovarian failure (POF) or premature ovarian insufficiency (POI) animal models, whereas little stem cell therapy has been practiced in clinical settings. The underlying molecular mechanism and safety of stem cell treatment in POI are not fully understood. In this study, we explored whether fetal mesenchymal stem cells (fMSCs) from the liver restore ovarian function and whether melatonin membrane receptor 1 (MT1) acts as a regulator for treating POI disease.

Methods: We designed an in vivo model (chemotherapy-induced ovary damage) and an in vitro model (human ovarian granulosa cells (hGCs)) to understand the efficacy and molecular cues of fMSC treatment of POI. Follicle development was observed by H&E staining. The concentration of sex hormones in serum (E2, AMH, and FSH) and the concentration of oxidative and antioxidative metabolites and the enzymes MDA, SOD, CAT, LDH, GR, and GPx were measured by ELISA. Flow cytometry (FACS) was employed to detect the percentages of ROS and proliferation rates. mRNA and protein expression of antiapoptotic genes (SURVIVIN and BCL2), apoptotic genes (CASPASE-3 and CASPASE-9), and MT1 and its downstream genes (JNK1, PCNA, AMPK) were tested by qPCR and western blotting. MT1 siRNA and related antagonists were used to assess the mechanism.

Results: fMSC treatment prevented cyclophosphamide (CTX)-induced follicle loss and recovered sex hormone levels. Additionally, fMSCs significantly decreased oxidative damage, increased oxidative protection, improved antiapoptotic effects, and inhibited apoptotic genes in vivo and in vitro. Furthermore, fMSCs also upregulated MT1, JNK1, PCNA, and AMPK at the mRNA and protein levels. With MT1 knockdown or antagonist treatment in normal hGCs, the protein expression of JNK1, PCNA, and AMPK and the percentage of proliferation were impaired.

Conclusions: fMSCs might play a crucial role in mediating follicular development in the POI mouse model and stimulating the activity of POI hGCs by targeting MT1.

Keywords: Fetal mesenchymal stem cells; MT1; Premature ovarian insufficiency; Reactive oxygen species.

Fig. 1

Characteristics of fMSCs. a Phenotypes of fMSC differentiation into adipocytes, chondrocytes, and osteocytes by light microscopy. b Immunophenotypes of fMSCs by flow cytometry. c Western blot analyses of Oct4, Nanog, and Rex-1 in fMSCs. fMSCs fetal mesenchymal stem cells

Fig. 2

Effects of fMSCs on follicle numbers and sex hormone levels in the POI mouse model. a Number of antral follicles at 8 weeks was calculated in NG, POI, and fMSC treatment groups. b Number of total follicles at 8 weeks was calculated in NG, POI, and fMSC treatment groups. c ELISA analysis of E2 levels was assessed at 0, 1, 2, 3, 4, and 8 weeks in NG, POI, and fMSC treatment groups. d ELISA analysis of AMH levels was assessed at 0, 1, 2, 3, 4, and 8 weeks in NG, POI, and fMSC treatment groups. e ELISA analysis of FSH levels at 0, 1, 2, 3, 4, and 8 weeks was assessed in NG, POI, and fMSC treatment groups. Data are represented as the mean ± SD. ***p < 0.001 (compared with the POI group)

Fig. 3

fMSCs decreased oxidative damage, increased oxidative protection, improved anti-apoptosis rates, and inhibited apoptosis in POI hGCs. a fMSCs were seeded on the upper coculture inserts, while POI hGCs were seeded on the bottom of a six-well culture plate. b FACS analysis of ROS expression at 8 weeks in different treatment groups. c–h ELISA analysis of MDA, SOD, CAT, LDH, GR, and GPx levels at 8 weeks in different treatment groups. i qPCR analysis of SURVIVIN and BCL2 in different treatment groups. j qPCR analysis of CASPASE3 and CASPASE9 in different treatment groups. k Western blot analysis of SURVIVIN, BCL2, CASPASE3, and CASPASE9 in different treatment groups. Data are represented as the mean ± SD. ***p < 0.001 (compared with the NG group)

Fig. 4

fMSCs decreased oxidative damage, increased oxidative protection, improved anti-apoptosis rates, and inhibited apoptosis in the POI mouse model. a fMSCs were injected into the POI mouse model by tail intravenous. b FACS analysis of ROS expression at 2 weeks in different treatment groups. c–h ELISA analysis of MDA, SOD, CAT, LDH, GR, and GPx levels at 8 weeks in different treatment groups. i qPCR detection of SURVIVIN and BCL2 levels in different treatment groups. j qPCR analysis of CASPASE-3 and CASPASE-9 levels in different treatment groups. k Western blot analysis of SURVIVIN, BCL2, CASPASE-3, and CASPASE-9 in different treatment groups. Data are represented as the mean ± SD. ***p < 0.001 (compared with the NG group)

Fig. 5

fMSCs upregulated MT1, JNK1, PCNA, and AMPK in vitro and in vivo. a qPCR analysis of MT1, JNK1, PCNA, and AMPK in different treatment groups in vitro. b Western blot analysis of MT1, JNK1, PCNA, and AMPK in different treatment groups in vitro. c qPCR detection of MT1, JNK1, PCNA, and AMPK in different treatment groups in vivo. d Western blot detection of MT1, JNK1, PCNA, and AMPK in different treatment groups in vivo

Fig. 6

fMSCs restored POI disease by targeting MT1. a Western blot analysis of MT1 in hGCs-MT1^KD cells. b Western blot analysis of JNK1, PCNA, and AMPK expression levels in different treatment groups. c Western blot assessment of MT1 after administration of the MT1 antagonist luzindole at different concentrations (10 μM, 20 μM, 40 μM, and 80 μM) in normal hGCs. d Western blot assessment of JNK1, PCNA, and AMPK in different treatment groups (luzindole, 40 μM). e KI67 expression levels were evaluated in different treatment groups by FACS. Data are represented as the mean ± SD. ***p < 0.05 (compared with the MT1^KD group)

Fig. 7

Proposed model for fMSCs improved ovarian function of POI mice through regulating MT1 pathway