The Paracrine Effect of Transplanted Human Amniotic Epithelial Cells on Ovarian Function Improvement in a Mouse Model of Chemotherapy-Induced Primary Ovarian Insufficiency

Abstract

Human amnion epithelial cells (hAECs) transplantation via tail vein has been reported to rescue ovarian function in mice with chemotherapy-induced primary ovarian insufficiency (POI). To test whether intraperitoneally transplanted hAECs could induce therapeutic effect and to characterize the paracrine effect of transplanted hAECs, we utilized a chemotherapy induced mice model of POI and investigated the ability of hAECs and conditioned medium collected from cultured hAECs (hAECs-CM) to restore ovarian function. We found that transplantation of hAECs or hAECs-CM either 24 hours or 7 days after chemotherapy could increase follicle numbers and partly restore fertility. By PCR analysis of recipient mice ovaries, the presence of SRY gene was only detected in mice transplanted with male hAECs 24 hours following chemotherapy. Further, the gene expression level of VEGFR1 and VEGFR2 in the ovaries decreased, although VEGFA increased 2 weeks after chemotherapy. After treatment with hAECs or hAEC-CM, the expression of both VEGFR1 and VEGFR2 increased, consistent with the immunohistochemical analysis. In addition, both hAECs and hAECs-CM treatment enhanced angiogenesis in the ovaries. The results suggested that hAECs-CM, like hAECs, could partly restore ovarian function, and the therapeutic function of intraperitoneally transplanted hAECs was mainly induced by paracrine-mediated ovarian protection and angiogenesis.

Figures

Figure 1

Identification and characterization of isolated hAECs. (a) The representative characteristics of hAECs under light microscope. (b) Expression of Oct-4, Nanog, and Sox2 (relative to a beta-actin internal control) in cultured hAECs by real-time PCR analysis. The results presented were the average values from five different donors. (c) The analysis of epithelial and mesenchymal markers expression (relative to a beta-actin internal control) in isolated hAECs by real-time PCR. The results presented were the average values from five different donors. (d) Immunofluorescence detection of Cytokeratin 18, CD34, and Vimentin in cultured hAECs. Scale bar: 200 μm.

Figure 2

The effect of human hAECs and hAECs-CM administration on mice body weight and the detection of transplanted hAECs. (a) Weight of mice treated with hAECs and hAECs-CM 24 hours after chemotherapy. The weight of Bu/Cy-treated administrated mice decreased significantly 7 days after chemotherapy when compared with mice in the control group (P < 0.01). Mice of hAECs- and hAECs-CM-treated groups weighed more compared with Bu/Cy mice treated with Bu/Cy administrated at any time point, although the differences were not significant. (b) The weight of mice that received Bu/Cy treatment and then an IP injection of hAECs or hAECs-CM 7 days later. (c) A representative gel electrophoresis of detection of the SRY gene in female mice ovaries treated with male hAECs by a simultaneous nested PCR analysis. The nested amplified product of the SRY sequence on the Y chromosome is 198 bp in length.

Figure 3

Histological analysis of mice ovaries and the follicle number count after treatment with hAECs or hAECs-CM. (a) Ovary tissues from each group were stained with hematoxylin and eosin. Numbers of oocyte-containing follicles at all stages were classified and counted in every fifth section. The primordial follicles (b), primary follicles (c), secondary follicles (d), and antral follicles (e) were identified and calculated. Data are shown as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar: 200 μm (Panels (a)(A)) and 50 μm (Panels (a)(B)), respectively.

Figure 4

Intraperitoneal injection of hAECs and hAECs-CM could partly restore fertility in mice treated with chemotherapy. The litter size per pregnancy was recorded. Data represent means ± SEM, ∗P < 0.05, ∗∗∗P < 0.001.

Figure 5

Real-time PCR analysis for VEGFR1, VEGFR2, and VEGFA expression in mice ovaries of different groups 2 weeks after treatment. ∗P < 0.05, ∗∗P < 0.01.

Figure 6

Immunohistochemical analysis for VEGFR1 expression in mice ovaries of different groups 2 weeks after treatment; ovarian sections with no primary antibody were served as negative controls. (a) control group; (b) Bu/Cy-treated group; (c) Bu/Cy + hAECs (24 h) group; (d) Bu/Cy + hAECs-CM (24 h) group; (e) Bu/Cy + hAECs (7 d) group; (f) Bu/Cy + hAECs-CM (7 d) group; (g) negative control. Scale bar: 100 μm.

Figure 7

Immunohistochemical analysis for VEGFR2 expression in mice ovaries of different groups 2 weeks after treatment; ovarian sections with no primary antibody were served as negative controls. (a) control group; (b) Bu/Cy administrated group; (c) Bu/Cy + hAECs (24 h) group; (d) Bu/Cy + hAECs-CM (24 h) group; (e) Bu/Cy + hAECs (7 d) group; (f) Bu/Cy + hAECs-CM (7 d) group; (g) negative control. Scale bar: 100 μm.

Figure 8

Immunohistochemical analysis for VEGFA expression in mice ovaries of different groups 2 weeks after treatment; ovarian sections with no primary antibody were served as negative controls. (a) control group; (b) Bu/Cy administrated group; (c) Bu/Cy + hAECs (24 h) group; (d) Bu/Cy + hAECs-CM (24 h) group; (e) Bu/Cy + hAECs (7 d) group; (f) Bu/Cy + hAECs-CM (7 d) group; (g) negative control. Scale bar: 100 μm.

Figure 9

Angiogenesis on Bu/Cy-treated ovaries after intraperitoneal injection of hAECs or hAECs-CM. Immunohistochemistry for CD34 ((a) and (c)) and microvessel density ((b) and (d)) were examined on ovaries obtained 2 weeks ((a) and (b)) or 1 month after treatment ((c) and (d)). Data were given as mean ± SEM. (A) control group; (B) Bu/Cy administrated group; (C) Bu/Cy + hAECs (24 h) group; (D) Bu/Cy + hAECs-CM (24 h) group; (E) Bu/Cy + hAECs (7 d) group; (F) Bu/Cy + hAECs-CM (7 d) group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar: 100 μm.