Adipose-derived stem cells improved mouse ovary function after chemotherapy-induced ovary failure

Min Sun, Shufang Wang, Yi Li, Ling Yu, Fang Gu, Changyong Wang, Yuanqing Yao, Min Sun, Shufang Wang, Yi Li, Ling Yu, Fang Gu, Changyong Wang, Yuanqing Yao

Abstract

Introduction: Young patients receiving chemotherapy occasionally face infertility and premature ovarian failure (POF). Numerous investigations reported that adipose-derived stem cells (ADSCs) transplantation could ameliorate the structure and function of injured tissues. The aim of this study was to explore the therapeutic efficacy of ADSC transplantation for chemotherapy-induced ovarian damage.

Methods: Female mice were injected intraperitoneally with 50 mg/kg cyclophosphamide (CTX). After 15 consecutive days of injection, ADSCs were transplanted either directly into bilateral ovaries or via intravenous injection, and the ovaries were excised after either 1 week or 1 month of treatment. The follicles were counted and categorized, and ovarian histologic sections were stained for TUNEL. Ovarian function was evaluated by monitoring ovulation. ADSC tracking, microarray analyses, and real-time polymerase chain reaction (PCR) were used to assess the inner mechanism of injury and repair.

Results: The ovarian function of mice exposed to CTX injection improved after ADSC transplantation. The population of follicles at different stages and ovulation significantly increased after the treatment. Immunofluorescence revealed reduced TUNEL staining. The tracking of ADSCs revealed that these cells did not directly differentiate into the follicle component. Microarray analyses indicated that changes in different groups of genes might affect follicle formation or ovulation.

Conclusions: ADSC transplantation improved ovarian function. Our results suggest a potential mechanism for ADSC therapy.

Figures

Figure 1
Figure 1
The isolation and identification of ADSCs. (A) The ADSCs exhibited typical fibroblastic morphology. (B) FCM analysis of ADSCs. The cells were positive for CD29, CD44, and CD90 and negative for CD31, CD34, and CD45. (C) ADSCs differentiate into osteoblasts and adipocytes. Scale bars: 100 μm.
Figure 2
Figure 2
The follicle number increased after transplantation. (A) Hematoxylin and eosin staining. (a) Most of the follicles of WT and ADSCs were secondary and antral follicles. The population of follicles decreased significantly after injection with CTX (b) and recovered after the ADSCs both intravenous and in situ therapy (c, d). The POF model mouse had fewer follicles and most of them were primary and secondary follicles or had no function. (B) The number of follicles after one week. (C) The number of follicles after 1 month. Scale bars: 200 μm. (*P < 0.05 in situ versus POF group; #P < 0.05 intravenous versus POF group).
Figure 3
Figure 3
The ovulation increased after therapy. (A) The oocytes of the WT group. (B) The oocytes in the POF model exhibited abnormal morphology and number. (C) The oocytes of the intravenous group. (D) The oocytes of the in situ group. (E, F) The population of oocytes increased after 1 week and 1 month of ADSC therapy. (*P < 0.05 intravenous versus the POF group, #P < 0.05 in situ versus the POF group).
Figure 4
Figure 4
The apoptosis of ovaries reduced after ADSC therapy. The ovaries of WT group (A, E), POF group (B, F), the intravenous group (C, G), and the in situ group (D, H). The green stain indicates TUNEL-positive GCs. The blue stain indicates the nucleus. Data from one week demonstrate that apoptosis was attenuated by the transplantation of ADSCs. (I) The number of apoptotic GCs decreased after 1 week of ADSC therapy. Scale bars: 100 μm (*P < 0.05 intravenous versus POF group, #P < 0.05 in situ versus POF group).
Figure 5
Figure 5
ADSCs tracking in vivo. (A) GFP (+)-ADSCs under a light microscope. (B) GFP (+)-ADSCs under a fluorescence microscope. (C, E) One week and 1 month after transplantation, ADSCs can be traced in the intravenous group. (D, F) One week and 1 month after transplantation, ADSCs can be traced in the in situ group. (G) ADSCs can not be traced in WT group. Scale bars: 100 μm.
Figure 6
Figure 6
Transplanted ADSCs were not formed into oocytes. (A) Oocytes and cumulus cells from donor ovaries showed green fluorescence. (B, C) Oocytes and cumulus cells from recipient ovaries did not show green fluorescence. Scale bars: 100 μm.
Figure 7
Figure 7
Microarray and real-time PCR analysis. (A) Gene expression in the intravenous group versus the WT group. (B) Gene expression in the intravenous group versus the WT group. (C) Gene expression in the in situ group versus the WT group. (D) Relative expression of genes. Angpt1, Onecut2, and Zcchc11 were upregulated, and Cxcr4 was downregulated; these results were in accordance with the microarray result.

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