hPMSC transplantation restoring ovarian function in premature ovarian failure mice is associated with change of Th17/Tc17 and Th17/Treg cell ratios through the PI3K/Akt signal pathway

Abstract

Background: Human placenta-derived mesenchymal stem cell (hPMSC) transplantation has been demonstrated to be an effective way of recovering ovarian function in mice with autoimmune induced premature ovarian failure (POF). But the exact mechanism remains unclear. The goal of the present study is to investigate the role of immune factors (T-helper 17 (Th17), cytotoxic T (Tc17) and regulatory T (Treg) cells) in the recovery of ovarian function and whether the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway is involved in the regulation.

Methods: The inhibitor of PI3K/Akt was administered to observe its effect on ovarian function recovery and immune regulation. Serum levels of estradiol (E₂), follicle stimulation hormone (FSH), luteinizing hormone (LH) and anti-Müllerian hormone (AMH)) and anti-Zona pellucida antibody (AZPAb) were measured by ELISA to evaluate ovarian function. The morphological changes of ovaries were observed by HE staining. Apoptosis of granular cells (GCs) was determined by detecting the expression of capase-3. Expression of p-Akt protein was detected by immunohistochemistry and western blot assay in ovarian tissues. The MTT assay was performed to assess GC proliferation. GC apoptosis was performed using flow cytometry analysis. Percentages of Th17, Tc17 and Treg cells were detected by flow cytometry. Expression of interleukin (IL)-17 in serum was measured by ELISA.

Results: LY294002 administration decreased serum levels of E₂ and AMH, while the levels of FSH, LH and AZPAb in serum were increased compared with mice in the hPMSC transplantation group. The ovarian morphology presented as atrophy and fibrosis, with functional follicles exhausted. The expression of p-Akt in ovarian tissue was significantly decreased. Also, LY294002 administration significantly decreased proliferation and increased cell apoptosis in GCs, and for immune factors the ratios of Th17/Tc17 and Th17/Treg cells were significantly increased, as well as the serum levels of IL-17.

Conclusions: Our data suggest that the PI3K/Akt signal pathway is involved in the recovery of ovarian function by changing the ratios of Th17/ Tc17 and Th17/Treg cells in POF mice following hPMSC transplantation.

Keywords: Akt; Human placenta-derived mesenchymal stem cells; Immune factors; PI3K; Premature ovarian failure.

Fig. 1

Cell surface markers, morphology and pluripotency of hPMSCs and GCs. Green histograms represent isotype control staining; blue histograms represent expression of indicated cell surface marker (a). hPMSCs cultured under conditions for differentiation into osteoblasts or adipogenesis (b–d). Cultured hPMSCs shows fibroblast-like morphology (b, ×200). Osteoblasts are displayed by Alizarin Red staining; darker red staining indicates calcium deposition (c, ×200). Adipogenesis displayed by accumulation of neutral lipid vacuoles stained with Oil Red O (d, ×200). Morphology and phenotypes of GCs (e–h). GCs shown as spindle-shaped morphology (e, ×200). DAPI staining (blue fluorescence) indicates the GC nucleus (f, ×400). FSHR-positive expression identified under Dylight 549 (red fluorescence) (g, ×400). Double-labeled staining (blue and red fluorescence) cells defined as FSHR-positive expression in GCs (h, ×400). FITC Annexin-V-fluorescein isothiocyanate, PE phycoerythrin

Fig. 2

Effects of LY294002 on the serum levels of E₂, FSH, LH, AMH, AZPAb and IL-17 in POF mice with or without hPMSC transplantation. a Estradiol (E₂) release. b Follicle stimulation hormone (FSH) release. c Luteinizing hormone (LH) release. d Anti-Müllerian hormone (AMH) release. e Anti-Zona pellucida antibody (AZPAb) release. f Interleukin (IL)-17 release. Data presented as mean ± SD. ^**P < 0.01, ^***P < 0.001 vs POF group. Groups: C control, M POF, T POF + hPMSCs, L POF + hPMSCs + LY294002, D POF + hPMSCs + DMSO. POF premature ovarian failure, hPMSC human placenta-derive mesenchymal stem cell, DMSO dimethylsulfoxide

Fig. 3

Histopathological examination of ovarian tissues. Photomicrographs (100×) show hematoxylin and eosin stained ovaries. (A) Control group (C group); four types of ovarian follicles observed (a–d). (B) POF group (M group). (C) POF + hPMSCs group (T group). (D) POF + hPMSCs + LY294002 group (L group). (E) POF + hPMSCs + DMSO group (D group). (F) Quantitation on follicle count from ovaries in mice of the five groups. Data presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs POF group. Bar scale = 200 μm. POF premature ovarian failure, hPMSC human placenta-derive mesenchymal stem cell, DMSO dimethylsulfoxide

Fig. 4

IHC analysis on PI3K, Akt, p-Akt and capase-3 in ovarian tissue of mice. Photomicrographs (400×) show hematoxylin and DAB-stained ovaries. (a1-d1) Control group (C group). (a2-d2) POF group (M group). (a3-d3) POF + hPMSCs group (T group). (a4-d4) POF + hPMSCs + LY294002 group (L group). (a5-d5) POF + hPMSCs + DMSO group (D group). The statistical charts of the four kinds of cytokines expression in the five groups (a6-d6). Brown in cytoplasm indicates positive expression of the aimed protein. Blue represents cell nuclear staining. ^**P < 0.01, ^***P < 0.001 vs POF group. Bar scale = 50 μm. POF premature ovarian failure, hPMSC human placenta-derive mesenchymal stem cell, DMSO dimethylsulfoxide, PI3K phosphatidylinositol 3-kinase

Fig. 5

AKT expression in ovarian tissues by western blot analysis. Akt and p-Akt protein expression (a) and quantification of Akt (b) and p-Akt (c) protein expression. Values expressed as mean ± SD. ^**P < 0.01, ^***P < 0.001 vs POF group. Groups: C control, M POF, T POF + hPMSCs, L POF + hPMSCs + LY294002, D POF + hPMSCs + DMSO. POF premature ovarian failure, hPMSC human placenta-derive mesenchymal stem cell, DMSO dimethylsulfoxide

Fig. 6

Effect of LY294002 on GC proliferation and apoptosis when cocultured with the supernatant of hPMSCs. The tendency charts of GC proliferation cocultured with 50 μl (a) or 100 μl (b) supernatant of hPMSCs. The statistical charts of GC proliferation cocultured with 50 μl (c) or 100 μl (d) supernatant of hPMSCs. e Representative flow cytometric plot for GCs apoptosis in different condition, and the percentage of cells in the upper right quadrant indicates late apoptotic cells. f The statistical chart of GCs apoptosis in all groups. Values expressed as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs POF group. GC granular cell, hPMSC human placenta-derive mesenchymal stem cell, DMSO dimethylsulfoxide, FITC Annexin-V-fluorescein isothiocyanate, PE phycoerythrin, OD optical density

Fig. 7

Effects of LY294002 on ratios of Th17/Tc17 and Th17/Treg cells. (a–c) Representative flow cytometric plots for Th17, Tc17 and Treg cell subset acquisition isolated from spleens, respectively. d Th17 cell population comparison among the groups. e Ratios of Th17/Tc17 comparison among the groups. f Treg cell population comparison among the groups. g Ratios of Th17/Treg comparison among the groups. Data expressed as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs POF group. Groups: C control, M POF, T POF + hPMSCs, L POF + hPMSCs + LY294002, D POF + hPMSCs + DMSO. POF premature ovarian failure, hPMSC human placenta-derive mesenchymal stem cell, DMSO dimethylsulfoxide, FITC Annexin-V-fluorescein isothiocyanate, PE phycoerythrin, IL interleukin