Heat shock pretreatment of mesenchymal stem cells for inhibiting the apoptosis of ovarian granulosa cells enhanced the repair effect on chemotherapy-induced premature ovarian failure

Xiaoying Chen, Qing Wang, Xinran Li, Qingru Wang, Jiaxin Xie, Xiafei Fu, Xiaoying Chen, Qing Wang, Xinran Li, Qingru Wang, Jiaxin Xie, Xiafei Fu

Abstract

Background: Premature ovarian failure (POF) is a severe complication associated with chemotherapy for female patients of childbearing age. A previous study has shown that bone marrow-derived mesenchymal stem cells (MSCs) can partially repair the damaged ovarian structure and function following chemotherapy. Heat shock (HS) is a pretreatment to enhance cell survival. The present study aimed to demonstrate the repair effect and potential working mechanism of HS MSCs on chemotherapy-induced POF.

Methods: Rat MSCs were isolated, cultured and identified. At 24 h, 48 h and 72 h after different strengths of HS pretreatment for 30 min, 1 h, 2 h and 3 h, apoptosis of MSCs was detected to determine the optimal conditions. Apoptosis and cell proliferation changes of MSCs were detected under the optimal conditions of HS. Apoptosis of HS preconditioned MSCs was detected after adding phosphamide mustard (PM) to mimic the microenvironment under chemotherapy. Rat granulosa cells (GCs) were isolated and cultured. PM was added and apoptosis of GCs was detected after coculture with the pretreated MSCs. The rat model of chemotherapy-induced POF was established and the pretreated MSCs were injected into bilateral ovaries. Ovarian structure and endocrine function were evaluated by ovary weight, follicle count, estrous cycle and sex hormone levels. Apoptosis of GCs was detected by TUNEL assay.

Results: The apoptosis rate of MSCs with 1 h of HS pretreatment decreased significantly, so 1 h was considered the optimal duration. Under this condition, the reduction in the apoptosis rate persisted until 120 h after the pretreatment and cell proliferation was accelerated. After HS pretreatment, MSCs displayed an increased tolerance to microenvironment under chemotherapy. After coculture with the HS-pretreated MSCs, PM-induced apoptosis of GCs decreased. Injection of the pretreated MSCs into the rat ovaries caused an increase in ovary weight and the number of follicles at different stages of estradiol levels, and a decrease in follicle stimulating hormone levels and apoptosis of GCs in the POF model.

Conclusion: HS pretreatment enhanced the repair effect of MSCs on chemotherapy-induced POF. The reason for this may be the further vitality enhancement of MSCs, which led to a greater inhibition of apoptosis of GCs.

Keywords: Bone marrow-derived mesenchymal stem cells; Chemotherapy; Heat shock; Premature ovarian failure.

Conflict of interest statement

Ethics approval

All experiments in the present study were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 86–23, revised 1985). All protocols in the present study were approved by the Animal Care and Research Committee of Southern Medical University, Guangzhou, China (Permit Number: L2016039). All surgeries and sacrifices were performed under chloral hydrate anesthesia. Every effort was made to minimize animal suffering.

Consent for publication

All authors agree to publish this manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Rat bone marrow mesenchymal stem cells and CM-Dil-labeled MSCs. a Primary MSCs at 6 days (white light ×50). b Primary MSCs at 6 days (white light ×100). c Suspended MSCs labeled with 4 μg/ml CM-Dil (white light × 200). d Suspended MSCs labeled with 4 μg/ml CM-Dil (fluorescence ×200)
Fig. 2
Fig. 2
Optimal condition of heat shock pretreatment for MSCs. a–e Apoptosis of MSCs at 48 h after HS pretreatment detected by flow cytometry: a normal group, b 30-min group, c 1-h group; d 2-h group; e 3-h group. f Apoptosis of MSCs at 24 h, 48 h and 72 h after HS pretreatment. *P < 0.05, compared with normal group. MSC mesenchymal stem cell
Fig. 3
Fig. 3
Effect of heat shock preconditioning on biological characteristics of MSCs. a Apoptosis rate of MSCs at 96 h, 120 h, 180 h and 240 h after HS at 42 °C for 1 h. Detected proliferative ability of MSCs with CCK-8. b Growth curve of HS MSCs and untreated MSCs. Flow cytometry to detect cell cycle by propidium iodide (PI) method at 48 h after HS. c Representative data for normal MSCs. d Representative data for HS MSCs. *P < 0.05, compared with normal group. HS heat shock, MSC mesenchymal stem cell
Fig. 4
Fig. 4
Apoptosis of MSCs and ovarian granulosa cells in each group. a Apoptosis of pretreated MSCs in local microenvironment under chemotherapy by flow cytometry. HS group. b Apoptosis rates of GCs in normal group, PM group, MSCs group and HS group by flow cytometry. HS group. *P < 0.05, compared with MSCs group. HS heat shock, MSC mesenchymal stem cell, PM phosphamide mustard
Fig. 5
Fig. 5
Ovarian structure and ovarian weight in each group. a Normal group (×50). b Model group (×50). c Sham group (×50). d MSCs group (×50). e HS group (×50). Arrows indicate follicles in ovary. f Ovarian weight of each group. *P < 0.05, compared with MSCs group. D day, HS heat shock, MSC mesenchymal stem cell
Fig. 6
Fig. 6
Amount of follicles at different stages in each group. a Primordial follicles. b Primary follicles. c Secondary follicles. d Antral follicles. *P < 0.05, compared with MSCs group. D day, HS heat shock, MSC mesenchymal stem cell
Fig. 7
Fig. 7
Sex hormone levels in each group. a Estradiol (E2). b Follicle stimulating hormone (FSH). *P < 0.05,compared with MSCs group. D day, HS heat shock, MSC mesenchymal stem cell
Fig. 8
Fig. 8
Apoptosis of ovarian granulosa cells in each group. Apoptosis index of ovarian granulosa cells at 1 day (a) and 60 days (b) after cell transplantation. #P < 0.05, compared with normal group; *P < 0.05, compared with MSCs group. GC granulosa cell, HS heat shock, MSC mesenchymal stem cell

References

    1. Faubion SS, Kuhle CL, Shuster LT, Rocca WA. Long-term health consequences of premature or early menopause and considerations for management. Climacteric. 2015;18:483–491. doi: 10.3109/13697137.2015.1020484.
    1. Kort JD, Eisenberg ML, Millheiser LS, Westphal LM. Fertility issues in cancer survivorship. CA Cancer J Clin. 2014;64(2):118–134. doi: 10.3322/caac.21205.
    1. Fu X, He Y, Xie C, Liu W. Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage. Cytotherapy. 2008;10(4):353–363. doi: 10.1080/14653240802035926.
    1. Fu X, He Y, Wang X, Peng D, Chen X, Li X, et al. Overexpression of miR-21 in stem cells improves ovarian structure and function in rats with chemotherapy-induced ovarian damage by targeting PDCD4 and PTEN to inhibit granulosa cell apoptosis. Stem Cell Res Ther. 2017;8(1):187. doi: 10.1186/s13287-017-0641-z.
    1. Devine PJ, Sipes IG, Hoyer PB. Initiation of delayed ovotoxicity by in vitro and in vivo exposures of rat ovaries to 4-vinylcyclohexene diepoxide. Reprod Toxicol. 2004;19(1):71–77. doi: 10.1016/j.reprotox.2004.06.002.
    1. O'Donnell RL, Warner P, Lee RJ, Walker J, Bath LE, Kelnar CJ, et al. Physiological sex steroid replacement in premature ovarian failure: randomized crossover trial of effect on uterine volume, endometrial thickness and blood flow, compared with a standard regimen. Hum Reprod. 2012;27(4):1130–1138. doi: 10.1093/humrep/des004.
    1. Donnez J, Dolmans MM. Fertility preservation in women. Nat Rev Endocrinol. 2013;9(12):735–749. doi: 10.1038/nrendo.2013.205.
    1. Sun X, Su Y, He Y, Zhang J, Liu W, Zhang H, et al. New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators. Cell Cycle. 2015;14(5):721–731. doi: 10.1080/15384101.2014.995496.
    1. Trounson A, McDonald C. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell. 2015;17(1):11–22. doi: 10.1016/j.stem.2015.06.007.
    1. Wang F, Wang L, Yao X, Lai D, Guo L. Human amniotic epithelial cells can differentiate into granulosa cells and restore folliculogenesis in a mouse model of chemotherapy-induced premature ovarian failure. Stem Cell Res Ther. 2013;4(5):124. doi: 10.1186/scrt335.
    1. Liu T, Huang Y, Zhang J, Qin W, Chi H, Chen J, et al. Transplantation of human menstrual blood stem cells to treat premature ovarian failure in mouse model. Stem Cells Dev. 2014;23(13):1548–1557. doi: 10.1089/scd.2013.0371.
    1. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol. 2001;33(5):907–921. doi: 10.1006/jmcc.2001.1367.
    1. Haider H, Ashraf M. Strategies to promote donor cell survival: combining preconditioning approach with stem cell transplantation. J Mol Cell Cardiol. 2008;45(4):554–566. doi: 10.1016/j.yjmcc.2008.05.004.
    1. Sart S, Ma T, Li Y. Preconditioning stem cells for in vivo delivery. Biores Open Access. 2014;3(4):137–149. doi: 10.1089/biores.2014.0012.
    1. Choudhery MS, Badowski M, Muise A, Harris DT. Effect of mild heat stress on the proliferative and differentiative ability of human mesenchymal stromal cells. Cytotherapy. 2015;17(4):359–368. doi: 10.1016/j.jcyt.2014.11.003.
    1. Moloney TC, Hoban DB, Barry FP, Howard L, Dowd E. Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells. Protein Sci. 2012;21(6):904–909. doi: 10.1002/pro.2077.
    1. Tutar L, Tutar Y. Heat shock proteins; an overview. Curr Pharm Biotechnol. 2010;11(2):216–222. doi: 10.2174/138920110790909632.
    1. Jung-Hynes B, Ahmad N. SIRT1 controls circadian clock circuitry and promotes cell survival: a connection with age-related neoplasms. FASEB J. 2009;23(9):2803–9. doi: 10.1096/fj.09-129148.
    1. Chua KF, Mostoslavsky R, Lombard DB, Pang WW, Saito S, Franco S, et al. Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress. Cell Metab. 2005;2(1):67–76. doi: 10.1016/j.cmet.2005.06.007.
    1. Kim J, Jang SW, Park E, Oh M, Park S, Ko J. The role of heat shock protein 90 in migration and proliferation of vascular smooth muscle cells in the development of atherosclerosis. J Mol Cell Cardiol. 2014;72:157–167. doi: 10.1016/j.yjmcc.2014.03.008.
    1. Feng Y, Huang W, Meng W, Jegga AG, Wang Y, Cai W, et al. Heat shock improves Sca-1+ stem cell survival and directs ischemic cardiomyocytes toward a prosurvival phenotype via exosomal transfer: a critical role for HSF1/miR-34a/HSP70 pathway. Stem Cells. 2014;32(2):462–472. doi: 10.1002/stem.1571.
    1. Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol. 2001;3(9):839–843. doi: 10.1038/ncb0901-839.
    1. Qiao PF, Yao L, Zhang XC, Li GD, Wu DQ. Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy. World J Gastroenterol. 2015;21(45):12822–12834. doi: 10.3748/wjg.v21.i45.12822.
    1. Herberg S, Shi X, Johnson MH, Hamrick MW, Isales CM, Hill WD. Stromal cell-derived factor-1beta mediates cell survival through enhancing autophagy in bone marrow-derived mesenchymal stem cells. PLoS One. 2013;8(3):e58207. doi: 10.1371/journal.pone.0058207.
    1. Gao F, Hu X, Xie X, Liu X, Wang J. Heat shock protein 90 stimulates rat mesenchymal stem cell migration via PI3K/Akt and ERK1/2 pathways. Cell Biochem Biophys. 2015;71(1):481–489. doi: 10.1007/s12013-014-0228-6.
    1. Matsuda F, Inoue N, Manabe N, Ohkura S. Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells. J Reprod Dev. 2012;58(1):44–50. doi: 10.1262/jrd.2011-012.

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