Background and objective: Cisplatin is an alkylating agent that has
become a first-line therapy for some tumors. However, overproduction of reactive
oxygen species (ROS) by cisplatin can cause male infertility, which can affect
patients’ quality of life. Melatonin, which has the ability to provide resistance
against oxidation, is a potential therapy for male infertility caused by
Material and methods: Normal human spermatozoa samples were divided
into (i) control group incubated with physiological saline solution; (ii)
cisplatin group incubated with cisplatin; (iii) melatonin group incubated with
melatonin; and (iv) melatonin + cisplatin group incubated with melatonin and
cisplatin. Spermatozoa motility was measured using a computer-aided semen
analysis system. Additionally, we determined spermatozoa apoptosis through
terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The
mitochondrial membrane potential (
Cisplatin is one of several platinum-based anticancer agents and is used for the
treatment of many types of tumors, including solid tumors, ovarian, breast,
brain, kidney, testicular, and head and neck tumors, as well as leukemia .
However, cisplatin has severe side effects on male fertility, which can
remarkably affect patients’ quality of life . Infertility induced by cisplatin
is believed to be associated with the overproduction of reactive oxygen species
(ROS) and increased levels of hydroxyl radicals (OH
Sperm are known to possess a high content of polyunsaturated fatty acids, plasmalogens, and sphingomyelins, making sperm sensitive to oxidative stress . Besides, lipids in sperm are easily oxidized, which can cause severe damage to the sperm . Cisplatin can also induce a mitochondrial-dependent radical response, which can damage nuclear DNA, enhancing the cytotoxic effect. The dysfunction of mitochondria induced by cisplatin varies among cells, depending on the mitochondrial redox status, mitochondrial DNA integrity, as well as bioenergetic function of the cells . Mature human sperm have 22–28 mitochondria helically arranged around the axoneme of the sperm, which generate ROS and ATP to maintain sperm motility. These mitochondria are potential targets of cisplatin [7, 8]. Therefore, it is desirable to find an effective therapy to protect sperm against cisplatin.
Melatonin is mainly produced by the pineal gland and plays major role in stimulating antioxidant enzymes and scavenging free radicals [4, 9, 10]. In many cell types, the nuclear factor erythroid 2-related factor (Nrf2) functions as antioxidant factor through the regulation of downstream genes, especially heme oxygenase-1 (HO-1) . It has been reported that melatonin can promote the activation of Nfr2 and the expression of the antioxidant enzyme HO-1 . However, it remains unknown whether melatonin can protect sperm against cisplatin. Therefore, in the present study, we investigated the effects of melatonin on human sperm characteristics and biochemistry as related to oxidative stress and a mechanism for the prevention of damage caused by cisplatin.
Cisplatin and melatonin were purchased from Sigma (St. Louis, MO, USA).
Sixty-six ejaculated sperm samples with
Stock solutions of cisplatin dissolved in physiological saline were stored at
Sperm motility was analyzed using computer assisted sperm analysis (CASA) as
described previously . An aliquot of 10
For each sample, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed according to the manufacturer’s protocol (Vazyme, Nanjing, China) to determine the apoptotic sperms, and 200 sperms were counted in a defined area on every slide. Slides were observed using an LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany), driven by ZEN2009 software (Carl Zeiss, Oberkochen, Germany).
The fluorescent, lipophilic and cationic probe, JC-1, was used to measure the
ATP levels were measured using an ATP assay kit (Beyotime Institute of
Biotechnology, Shanghai, China) according to the manufacturer’s instructions.
Each sample was mixed with 20% trichloroacetic acid and 0.67% thiobarbituric
acid, and the mixture was incubated at 100
Proteins were extracted from the human sperms. The expression of Nrf2 and HO-1
was examined by western blot.
Statistical analysis was performed using SPSS 19.0 statistical software
(IBM Corp., Chicago, IL, USA). All values are presented as the mean
Different concentrations of cisplatin (25
Melatonin significantly restored human sperm motility. (a)
Sperms were incubated with different concentrations of cisplatin for 3 h. The
figure shows total sperm motility (a–c) and progressive motility. ***p
TUNEL staining was used for the evaluation of sperms apoptosis. We found that
the number of TUNEL-positive sperms in the melatonin + cisplatin group was higher
than that in the control group (p
Melatonin decreased cisplatin-induced spermatozoa apoptosis.
The effect of melatonin on spermatozoa apoptosis was investigated. Microscopic
images of human spermatozoa stained with terminal deoxynucleotidyl
transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling
(TUNEL) (a–d). Scale bar = 50
There were fewer sperms with low
Melatonin restored mitochondrial function. The mitochondria
functions of spermatozoa were measured, including (a–e) measurement of
mitochondrial membrane potential by a cytometry technique and (f) ATP production
by spermatozoa from different groups. Control: Spermatozoa were treated with
physiological saline; Cisplatin: Spermatozoa were treated with 250
As shown in Fig. 4, the levels of MDA and ROS in sperms were markedly increased in the cisplatin group compared those in the control group. However, the levels of MDA and SOD in the sperms were decreased in the melatonin + cisplatin group compared with the cisplatin group.
Melatonin ameliorated oxidative stress induced by cisplatin.
The effects of melatonin on sperm antioxidant levels and oxidative status were
investigated, including (a–e) SOD activity and (f) MDA activity. Control: Sperms
were treated with physiological saline; Cisplatin: Sperms were treated with 250
Our results showed that cisplatin exposure repressed the expression of the antioxidant pathway components Nrf2 and HO-1. Treatment with melatonin activated the Nrf2 and HO-1 expression reduced by cisplatin (Fig. 5a,b).
Melatonin improved antioxidant gene expression decreased by
cisplatin. (a) Nrf2 and HO-1 protein levels were analyzed by western blotting.
(b) Each data point was calculated from three triplicate groups and the data are
presented as the mean
Cisplatin is an effective anticancer drug, but it has demonstrated toxicity to the bladder , inner ear , peripheral nerves , and reproductive system . Melatonin is capable of attenuating testis toxicity induced by microwave radiation , 2-bromopropane , and busulfan ; thus, melatonin may be useful in preventing the effect of cisplatin-induced toxicity on fertility. Although cisplatin has been shown to induce damage in the early stage of spermatogenesis in human and animal models [21, 22], considering the difficulty of the application of melatonin in humans, we used ejaculated human sperm as our model to examine the effect of melatonin on fertility.
In our study, we demonstrated that cisplatin can adversely affect the motility of human sperm, which was consistent with previous reports . Furthermore, we showed that a low dose of melatonin markedly alleviated the observed decrease in the motility of human sperm caused by cisplatin. However, when the melatonin dose exceeded 100 mM, melatonin itself inhibited human sperm motility. The reasons for this effect remain unknown. Considering the existence of the blood-testis barrier and the severe toxicity of potent metabolites formed from cisplatin, 100 nM melatonin could be viewed as a reference for clinical treatment.
Mitochondria, which are arranged around the mid-piece axoneme of human sperm,
are the primary targets of drugs and the cause of human sperm death. The motility
of human sperm is mainly maintained by mitochondria, which have important role in
the generation of ATP as the molecular motor generating power for sperm flagellar
movement. To further understand the cause of reduced human sperm motility, we
The disruption of the
MDA is a major product of the lipid peroxidation of membrane polyunsaturated fatty acids. In the present study, cisplatin increased the MDA levels in human sperm compared with the control group, while these cisplatin-induced MDA increases were reduced by melatonin, further indicating that melatonin may be able to protect sperm from the effects of cisplatin.
In addition, we showed that melatonin upregulated the levels of the antioxidant factor Nrf2 and HO-1, indicating that melatonin might protect human sperm through activating Nrf2 and its downstream gene HO-1.
In summary, melatonin, as antioxidant, can protect against the cisplatin-induced toxicity of human sperm, providing a new strategy for treating clinical infertility. However, a lack of research into the effects of melatonin in the early stage of human spermatogenesis currently limits the use of melatonin in the treatment of cisplatin-induced testis toxicity in the clinic. However, the present study indicates that using melatonin may be a useful strategy in the prevention of cisplatin-induced testis toxicity.
ATP, adenosine triphosphate; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor; HO-1, heme oxygenase-1; DMSO, dimethyl sulfoxide; HTF, human tubal fluid; CASA, computerassisted sperm analysis; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; TCA, trichloroacetic acid; TBA, thiobarbituric acid; MDA, malondialdehyde; BTB, blood-testis barrier.
YS, ZW, and QZ designed the research study. BS performed the research. JJ collected samples. JX analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
This study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Donors were informed about the purpose of the study and their consent was obtained. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanjing Medical University ( KY 041).
We thank Liwen Bianji, Edanz Group (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
This research was funded by the National Natural Science Foundation of China (No. 81400758) and Science and technology development fund of Nanjing Medical University (NMUB2019048) for supporting our work.
The authors declare no conflict of interest.