Antioxidative effect of melatonin on cryopreserved ovarian tissue in mice
Introduction
The ovaries are important organs for female reproductive functions. Although the survival rate of cancer patients has improved with radiotherapy and chemotherapy, these cancer treatments may cause irreversible damage to the ovaries, and even cause premature ovarian insufficiency, loss of fertility and endocrine function [25]. Thus, the maintenance of long-term quality of life and fertility of cancer survivors has become a major concern. For adolescent cancer patients, cryopreservation of oocytes or embryos is not appropriate. Therefore, cryopreservation of ovarian tissues (OTs) has become an option for cancer survivors to preserve their fertility [22,25].
Cryopreservation and auto-transplantation of ovarian cortex have been widely used in the preservation and restoration of fertility in various species, including human [9]. To date, although more than 86 babies have been born worldwide through cryopreserved-thawed OTs, there are many unsolved problems [11]. For example, there is cryopreserved-thawed damage to OTs and high rates of intact follicle loss during cryopreservation [25]. In addition, reactive oxygen species (ROS) is produced during cryopreservation of OTs, which can destroy follicles and cause oxidative damage [19,28]. Studies have shown that ROS is one of the key factors affecting the cryopreservation effect of vitrification, which can induce apoptosis or dysfunction of cells, and ultimately lead to the decline of oocyte survival rate and development ability [7,16,18]. Therefore, addition of exogenous antioxidants in vitrified solution can reduce the production of ROS and the oxidative stress response [3,19,20].
Melatonin (MLT) is a neuroendocrine hormone secreted by the pineal gland. It is an effective antioxidant with a wide range of biological effects and can resist oxidative stress through a variety of mechanisms [3,19,28]. For example, MLT can be used as a scavenger of toxic oxygen derivatives and reduce the formation of ROS [19]. In addition, MLT has a variety of biological functions such as increasing gene expression or activity of other antioxidant enzymes, preventing oxidative stress damage, inhibiting lipid peroxidation and preventing DNA damage [3,19,27]. In recent years, the researches on the role of MLT in the reproductive system are increasing [3,19,27]. Our study has demonstrated interactions between NF-E2-related factor-2 (Nrf-2), heme oxygenase-1 (HO-1) and MLT, and it is suggested that MLT may have a role in the development of polycystic ovaries [28]. Recent evidence suggests that MLT has a protective effect on OTs [1,8]. However, the effect of MLT on cryopreservation of OTs is less studied.
In this study, we aim to investigate the role of MLT in protecting OTs from the negative effects of cryopreservation.
Section snippets
Ovarian collection and experimental design
OTs (n = 106) of adolescent SPF KM mice (7 weeks of age, certificate number: 211,002,300,043,372) were collected and randomly divided into fresh control group (n = 6) and experimental group (n = 100). The experimental group was subdivided into five groups (0 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1 mM) according to the concentrations of MLT in the vitrification solutions (n = 20 of each group). OTs of fresh control group were fixed in 4% paraformaldehyde while those of experimental group were washed
Morphological assessment of the fresh and cryopreserved-thawed OTs
A total of 3433 follicles from 930 histological sections of the fresh and five MLT-(un)treated groups were morphologically evaluated by HE staining. Compared with MLT-untreated group, the MLT-treated groups demonstrated a well preserved morphology of follicles, although there were areas of decreased stromal density or irregular morphology of follicles and oocytes in some samples (Fig. 1). Table 2 shows the number and proportion of the morphologically normal follicles in fresh and cryo-thawed
Discussion
The aim of our study is to evaluate the effects and mechanisms of MLT on viability, follicle classification, morphology, ROS production, and oxidative activities of OTs during cryopreservation.
Cryopreservation of OTs is challenging because several cell types in OTs are vulnerable to severe damage [4,15,24]. Previous studies have reported the beneficial effects of different antioxidants on cryopreservation of OTs, such as resveratrol EG and PrOH [16,25]. The evaluation of antioxidant protection
Conclusions
In summary, our study showed that MLT had multiple effects on cryopreserved-thawed OTs, including enhancing antioxidative enzyme activation, reducing the production of ROS, and inhibiting follicular cell apoptosis. Furthermore, 0.1 mM MLT had no cytotoxic effect on cryopreserved-thawed OTs. Therefore, MLT protects the quality of cryopreserved OTs by decreasing oxidative stress level and the optimal concentration is 0.1 mM.
Author contributions
Authors’ individual contributions: conceptualization, S·H·Y, L.Y·C; methodology, T.C·S, H·Y.L; investigation, X.C.L, T.C·S, and L.Y·C; data curation, L.N·S and M.W; writing-original draft preparation, T.C·S, H·Y.L, and X.C.L; writing-review and editing, T.C·S, Z.H·C, S·H·Y, and L.Y·C; funding acquisition, S·H·Y, and L.Y·C; X.C.L, T.C·S, and H·Y.L have contributed equally to this work. All authors have read and agreed to the published version of the manuscript.
Funding
This study was funded by the Hebei Higher Education Research Project (Grant No. QN2015121), Peking Post-doctoral Research Fund (EE2019-50), Peking University International Hospital Research Funds (No. YN2019QN13) and National Natural Science Foundation of China (81441133 and 81602318).
Declaration of competing interest
The authors have no conflicts of interest relevant to this article.
Acknowledgments
The authors would like to express sincere gratitude to Dr. Xiao Yu Li, Guangdong Medical University, for his support in data management and statistical analysis.
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