Human amniotic epithelial cells inhibit granulosa cell apoptosis induced by chemotherapy and restore the fertility

Abstract

Introduction: Premature ovarian failure and insufficiency are significant long-term side-effects of chemotherapy for female cancer patients. Recently, stem cell transplantation has been identified as a promising treatment for premature ovarian failure and insufficiency. We have previously demonstrated that human amniotic epithelial cells (hAECs) migrate into injured tissue and promote the recovery of ovarian function in chemoablated mice. However, the molecular mechanism guiding this process remains unclear.

Methods: To further investigate the effect of hAECs on chemotherapy-induced apoptosis, cultured primary hAECs were injected intravenously into mice treated with cyclophosphamide and busulphan. Apoptosis of granulosa cells was observed by TUNEL staining, and apoptosis-related gene expression was performed on ovarian tissue by real-time PCR and Western blot 7 days after hAEC transplantation. Additionally, the ovarian function and fertility of mice were assessed via counts of follicles and mating experiments at 4 weeks after hAEC transplantation.

Results: hAECs significantly inhibited tumor necrosis factor-alpha-mediated granulosa cell apoptosis induced by chemotherapeutics and reduced the inflammatory reaction in ovaries at 7 days after transplantation. In addition, 4 weeks after transplantation, hAECs promoted the development of follicles and increased the number of cumulus oocyte complexes in chemoablated mice. Furthermore, hAECs improved ovarian mass and increased the number of follicles compared to those of the chemoablated group, and hAEC transplantation partially rescued the fertility of chemoablated mice.

Conclusions: hAEC transplantation promotes ovarian function by inhibiting tumor necrosis factor-alpha-mediated cell apoptosis and reducing inflammation in chemotherapy-induced premature ovarian failure. These results suggest a potential molecular mechanism for the effective therapy of hAEC transplantation in chemotherapy-induced premature ovarian failure and insufficiency.

Figures

Fig. 1

Chemotherapy reduced body weight of mice and the number of ovarian follicles. A Bar graph illustrating the body weight of mice in the sham and chemoablated (Cy) groups. B H&E staining of ovaries in sham and treatment groups, 7 and 10 days post-induction. Blue arrow indicated primordial follicle; red arrow indicated primary follicle; black arrow indicated secondary follicle; white arrow indicated mature follicle in B-b. C Bar graph representing the number of follicles at different stages of development. Data represent means ± SEM; *p < 0.05 versus Sham. Scale bar = 500 μm (a, c and d), 200 μm in b. Sham sham group (n = 6), Cy-3days chemoablated 3-day group (n = 6), Cy-7days chemoablated 7-day group (n = 6), Cy-10days chemoablated 10-day group (n = 6)

Fig. 2

Chemotherapy-induced apoptosis of granulosa cells within developing follicle. A TUNEL-positive cells were observed in the ovary tissue at 7 days after chemotherapy. The red stain indicates TUNEL-positive granulosa cells. The blue DAPI stain indicates the cell nucleus. B Relative expression of Bcl2, Bax and TNF-α mRNA in ovarian tissue at 3, 7 and 10 days after chemotherapy. Data represent means ± SEM; *p < 0.05 versus Sham. Scale bar = 200 μm (a, b, c and d), 100 μm (e and f). Sham sham group (n = 6), Cy-3days chemoablated 3-day group (n = 6), Cy-7days chemoablated 7-day group (n = 6), Cy-10days chemoablated 10-day group (n = 6)

Fig. 3

hAEC transplantation reduced chemotherapy-induced inflammation in ovarian tissue. A Section of dissected hAECs illustrated a cobble stone-like epithelial cell morphology. B Relative expression of mRNA levels detected by real-time PCR in cultured hAECs. C Image of fixed ovarian tissue in different groups at 7 days after hAEC transplantation. D Bar graph demonstrated the ovarian weights in different groups. E H&E staining results displaying ovarian morphology in different groups at 7 days after hAEC transplantation. F Relative expression of inflammatory cytokine mRNA levels in injured ovarian tissue analyzed by real-time PCR at 7 days after hAEC transplantation. Data represent means ± SEM; *p < 0.05 versus Sham, #p < 0.05 versus Cy. Scale bar = 200 μm in (E). Cy chemoablated group (n = 6), Cy + hAECs hAEC-treated group (n = 6), Sham sham group (n = 4)

Fig. 4

hAEC transplantation inhibited chemotherapy-induced apoptosis in ovaries. A Relative mRNA expression of TNF-α, TRADD, FADD, caspase-3, Bcl2 and Bax was detected by real-time PCR in ovarian tissue at 7 days after hAECs transplantation. B Western blot results and corresponding densitometry analysis demonstrated that chemotherapy significantly increased activated caspase-3 protein expression at 7 days after hAEC transplantation. A significant reduction of activated caspase-3 protein expression in the hAEC-treated group compared with the chemoablated group. Data are mean ± SEM; *p < 0.05 versus Sham; #p < 0.05 versus Cy. Cy chemoablated group (n = 6 in A, n = 4 in B), Cy + hAECs hAEC-treated group (n = 6 in A, n = 4 in B), Sham sham group (n = 4 in both panels)

Fig. 5

hAEC transplantation increased the number of cumulus oocyte complexes (COC) in chemoablated mice. A–C Images of ovarian morphology followed superovulation in different groups 1 month after hAEC transplantation (white arrows). D–F The morphology of COC was observed under microscopy. G Bar graph representing the counting of COC in different groups. H–J H&E staining of ovaries followed superovulation was used to observed follicle development. Enlargements of outlined areas are shown in panels a–f. Mature follicles are indicated by the black arrow (a and e). Primary follicles are indicated by the black arrowhead (b, d and f). Follicular atresia is indicated by the white arrowhead (c). Data are mean ± SEM; *p < 0.05 versus Sham; #p < 0.05 versus Cy. Scale bars = 200 μm (H–J), 100 μm (a, c and e), 50 μm (b, d and f). Cy chemoablated group (n = 4), Cy + hAECs hAEC-treated group (n = 4), Sham sham group (n = 4)

Fig. 6

hAEC transplantation increased the number of ovarian follicles and restored the fertility of chemoablated mice. A Bar graph illustrating body weight of experimental and control groups following hAEC transplantation. B The ovarian weight in different groups at 28 days after hAEC transplantation. C Histological analysis of ovaries in different groups at 28 days after hAEC transplantation. D The number of different stage follicles counted at 28 days after hAEC transplantation. E Bar graph representing the number of pups per pregnancy at the end of mating. Data are mean ± SEM; *p < 0.05 versus Sham; #p < 0.05 versus Cy. Scale bars in c = 500 μm (a–c), 200 μm (d–f), 100 μm (g–i), 50 μm (j–l). Cy chemoablated group (n = 6; n = 10 in E), Cy + hAECs hAEC-treated group (n = 6; n = 10 in E), Sham sham group (n = 6; n = 5 in E)