Received: 24 February 2020

| Revised: 23 July 2020

| Accepted: 1 August 2020

DOI: 10.1111/jpi.12690

ORIGINAL ARTICLE

Melatonin protects mouse testes from palmitic acid-induced

lipotoxicity by attenuating oxidative stress and DNA damage in a
SIRT1-dependent manner

Dejun Xu1,2 | Lingbin Liu1 | Yongju Zhao1 | Li Yang2 | Jianyong Cheng2 |

Rongmao Hua2 | Zelin Zhang2 | Qingwang Li2

1
College of Animal Science and
Technology, Southwest University,
Chongqing, China
2
College of Animal Science and
Technology, Northwest A&F University,
Yangling, China
Correspondence
Qingwang Li, College of Animal Science
and Technology, Northwest A&F
University, No.3 Taicheng Road, Yangling
712100, China.
Email: 2015060124@nwafu.edu.cn
Funding information
Key Industry Innovation Chain of
Shaanxi Province, Grant/Award Number:
2018ZDCXL-NY-02-06; Ministry of
Agriculture Transgenic Major Projects,
Grant/Award Number: 2018ZX0801013B;
National Key Technology Support Program,
Grant/Award Number: 2015BAD03B04
Abstract
Palmitic acid (PA), the main component of dietary saturated fat, has been known
to increase in patients with obesity, and PA-induced lipotoxicity may contribute to
obesity-related male infertility. Melatonin has beneficial effects on reproductive pro-
cesses; however, the effect and the underlying molecular mechanism of melatonin's
involvement in PA-induced cytotoxicity in the testes are poorly understood. Our
findings showed that lipotoxicity was observed in mouse testes after long-term PA
treatment and that melatonin therapy restored spermatogenesis and fertility in these
males. Moreover, melatonin therapy suppressed PA-induced apoptosis by modu-
lating apoptosis-associated proteins such as Bcl2, Bax, C-Caspase3, C-Caspase12,
and CHOP in type B spermatogonial stem cells. Changes in the expression of endo-
plasmic reticulum (ER) stress markers (p-IRE1, p-PERK, ATF4) and intracellular
Ca2+ levels showed that melatonin relieved PA-induced ER stress. Mechanistically,
melatonin stimulated the expression and nuclear translocation of SIRT1 through its
receptors and prevented PA-induced ROS production and mitochondrial dysfunction
via SIRT1 signaling pathway. Furthermore, melatonin promoted SIRT1-mediated
p53 deacetylation, thereby relieving G2/M arrest in response to PA-stimulated DNA
damage. Collectively, these findings indicate that melatonin protects the testes from
PA-induced lipotoxicity through the activation of SIRT1, which alleviates oxidative
stress, ER stress, mitochondrial dysfunction, and DNA damage.

KEYWORDS
DNA damage, melatonin, oxidative stress, palmitic acid, SIRT1, testis

1 | IN T RO D U C T ION
It is well known that unhealthy obesity leads to an increased
risk of male infertility.1 Although the primary pathogenesis
of obesity-associated male infertility remains unclear, exces-
sive free fatty acids (FFAs) are recognized as a major cause of
this disease.2 In adipocytes, FFAs are stored and released for
energy homeostasis; however, in obese individuals, adipose
tissues exhibit excessive FFAs, which lead to a myriad of
cellular dysfunctions, which can ultimately trigger apoptotic
cell death in nonadipose cells in a process known as lipotox-
icity.3 Obesity is usually accompanied by dysregulated FFA

© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

J Pineal Res. 2020;69:e12690.  wileyonlinelibrary.com/journal/jpi | 1 of 20

https://doi.org/10.1111/jpi.12690
2 of 20
|    XU et al.
metabolism including elevated levels of saturated fatty acids,
and in the plasma, palmitic acid (PA) is the most common
type of saturated FFA.4 It has been reported that levels of
FFAs, including PA, are increased in patients with obesity.5-7
A clinical study also demonstrated that dietary intake of PA
was positively associated with asthenozoospermia, indicat-
ing an association between PA intake and male infertility.8
These findings have demonstrated that elevation in PA levels
may play an important role in obesity-related male infertility.
Further research indicates that excessive PA causes lipotoxic
effects, and this effect is mediated by beta-oxidation-induced
oxidative stress.4,9 Specifically, mammalian spermatozoa
are especially vulnerable to reactive oxygen species (ROS),
since they are rich in polyunsaturated fatty acids, which in
turn leads to male infertility.10 Therefore, it is necessary to
identify effective approaches to ameliorate PA-induced lipo-
toxicity in the testis.
N-acetyl-5-methoxytryptamine (melatonin), an endoge-
nous hormone mainly secreted by the pineal gland, is also
synthesized in reproduction-related tissues such as ovaries
and testes.11,12 It has various important physiological func-
tions, including regulation of inflammation,13 apoptosis,14
and metabolism.15 Recently, much attention has been given to
the role of melatonin in male infertility. Melatonin has been
shown to be involved in male reproduction by modulating
steroid hormone secretion16 as well as by being involved in
the proliferation of spermatogenic cells.17 Moreover, mela-
tonin protects the testis against hyperthermia, environmental
toxins, and drug-induced damage18-20 due to its lipophilic and
hydrophilic free radical scavenging characteristics.21 Indeed,
previous studies have demonstrated the beneficial effects of
melatonin treatment on testicular cells under pathological
conditions; however, it is unclear whether melatonin can pre-
vent obesity-associated male infertility, specifically in PA-
induced testicular damage.
SIRT1, a member of the conserved nicotinamide ade-
nine dinucleotide (NAD+)-dependent deacetylase family,
has been suggested to be involved in various biological func-
tions, including stress resistance, differentiation, and gene
silencing based on deacetylation of substrate proteins.22,23
Deacetylation of forkhead transcription factors of class O
(FOXO) by SIRT1 is involved in oxidative stress resistance.24
Similarly, SIRT1 has been shown to deacetylate and inacti-
vate p53 in response to DNA damage.25 Interestingly, recent
studies have shown that melatonin exerts biological effects
via a SIRT1-dependent mechanism in aging, inflammation,
and embryo development.26-28 Although evidence linking
melatonin to the role of SIRT1 has been reported, only a few
of the molecular mechanisms involved have been addressed.
It would be interesting to clarify the functional link between
SIRT1 and melatonin in response to PA-induced lipotoxicity.
In the present study, we investigated whether melatonin
can exert a protective effect on PA-induced cytotoxicity in
mouse testes and type B spermatogonial stem cells (GC-1).
Furthermore, we sought to identify the signal transduction
pathways of melatonin and the possible underlying mecha-
nisms involved in this cytoprotective action.
2 | M ATERIAL S AND M ETHOD S
2.1 | Chemicals
Melatonin (Cat#: 73-31-4) and ER stress antagonist 4-PBA
(Cat#: 1716-12-7) were purchased from Sigma-Aldrich,
whereas melatonin receptor antagonist luzindole (Cat#:
117946-91-5) was purchased from Santa Cruz Biotechnology,
Inc. SIRT1 antagonist EX527 (Cat#: S1541) and p38 MAPK
antagonist BMS-582949 (Cat#: S8124) were purchased
from Selleck chemicals. The antibodies used in this study
were as follows: anti-melatonin receptor 2 (Cat#: 41139),
anti-SIRT2 (Cat#: 32057), anti-SIRT3 (Cat#: 39196), anti-
Cleaved Caspase3 (Cat#: 29034), anti-Cleaved Caspase12
(Cat#: 24209), anti-CHOP (Cat#: 40744), anti-IRE1(Cat#:
45107), anti-pIRE1 (Ser724; Cat#: 13013), anti-PERK (Cat#:
33247), anti-p-PERK(Ser555; Cat#: 12887), anti-ATF4
(Cat#: 32007), anti-p38 MAPK(Cat#: 49379), anti-p-p38
MAPK (Thr180/Tyr182; Cat#: 11581), anti-Acetyl-FoxO1
(Cat#: 30033), and anti-Acetyl-p53 (Cat#: HW109) were
obtained from Signalway antibody LLC; anti-SIRT1 (Cat#:
60303-1-Ig), anti-Tubulin (Cat#: 66031-1-Ig), anti-PLZF
(Cat#: 66672-1-Ig) were obtained from ProteinTech Group,
Inc; anti-melatonin receptor 1 (bs-0027R), anti-ERK1/2
(Cat#: bs0022R), anti-p-ERK1/2 (Thr202/Tyr204; Cat#:
bs3016R), anti-FoxO1 (Cat#: bs-2537R), anti-MnSOD
(Cat#: bs-20668R), anti-PGC1α (Cat#: bs-1832R) were pur-
chased from Bioss; anti-Oct4 (Cat#: AF0249), anti-SOX2
(Cat#: AF8034), anti-SOX9 (Cat#: AF2329), anti-BCL2
(Cat#: AF6285), anti-Bax (Cat#: AF0054), anti-Nrf2 (Cat#:
AF7623), anti-p-Nrf2 (Ser40; Cat#: AF1609), anti-Catalase
(Cat#: AF0084), anti-PCNA (Cat#: AF1363), and anti-
Histone H3 (Cat#: AF0009) were obtained from Beyotime.
Unless otherwise indicated, the other chemicals were pur-
chased from Sigma-Aldrich.
2.2 | Animal experiments
All mice in this experiment were treated in accordance with
the Animal Research Institute Committee guidelines of
Northwest A&F University, China. C57BL/6 mice (6 weeks
old) were housed in wire cages under conditions of constant
temperature (25°C), humidity (70%), and lighting (12 hours
light/12 hours dark cycle), and were fed with a standard
laboratory rodent diet with water provided ad libitum during
this study. Melatonin was dissolved in ethanol and diluted in
XU et al.
normal saline solution to a final concentration of 5% etha-
nol. Palmitic acid (PA) was prepared in ethanol and diluted
in normal saline solution containing albumin (10%) for the
PA-BSA complex solution. The male mice were randomly
assigned into three groups: The PA group was injected intra-
peritoneally with PA at a dose of 100 mg/kg/d, the PA + me-
latonin group was injected intraperitoneally with melatonin
at a dose of 10 mg/kg/d before PA treatment, and the control
group followed the same regimen and received sham injec-
tions (without melatonin and PA). After 30 days of treatment,
the mice were sacrificed and the testicular tissues were col-
lected for analysis. The estrous cycles of female mice were
mated to males. There before, male mice were treated with
PA (100 mg/kg/d) with or without melatonin (10 mg/kg/d)
for 30 days. The pups were born approximately 22 days after
mating, and the pregnancy rates and number of offspring
were recorded and statistically analyzed.
2.3 | Assessment of sperm parameters
Epididymal sperm were obtained according to the method of
Mohammad et al.29 Briefly, a small piece of the cauda was
cut and incubated in 1 mL of prewarmed physiological saline
at room temperature for 5 minutes to allow the movement
of spermatozoa from epididymal to fluid. Sperm concentra-
tion was measured using Neubauer's chamber as previously
described.30 Sperm concentration data were expressed as
106 cells/mL. Furthermore, sperm motility was assessed by
counting motile and nonmotile sperm and was expressed as
the percentage of motile sperm of the total sperm count. For
sperm morphological analyses, the cauda epididymal sperm
were spread onto slides and were stained with Giemsa stain-
ing solution. Sperm with morphological abnormalities in the
head and flagellum were counted and expressed as percent-
age of the total sperm count.
2.4 | Cell culture and treatment
The type A spermatogonial stem cells (SSCs, C18-4) were
cultured in Dulbecco's modified Eagle's medium/nutrient
mixture F12 (DMEM/F12) supplemented with 10% fetal bo-
vine serum (FBS), 2 mmol/L L-glutamine, and 1% albumin
(for PA-BSA complex solution) at 37°C in a humidified at-
mosphere containing 5% CO2. Type B SSCs (GC-1) were
cultured in DMEM supplemented with 10% FBS, 2 mmol/L
L-glutamine, and 1% albumin. Leydig cells (MLTC1) were
cultured in 1640 medium supplemented with 10% FBS,
2 mmol/L L-glutamine, and 1% albumin. According to pre-
vious reports31-34 and viability assays, the final concentra-
tions of PA (100 μmol/L), melatonin (1 μmol/L), EX527
(2 μmol/L), 4-PBA (250 μmol/L), luzindole (10 μmol/L),
  
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and BMS-582949 (10 μmol/L) were used in this study. In all
experiments, the concentrations of ethanol and DMSO were
diluted to below 1% (v/v).
2.5 | Histology analysis
Testicular tissues were collected and fixed in 4% (w/v) para-
formaldehyde solution. After washing with PBS, samples
were embedded in paraffin blocks; afterward, sections (5 μm)
were cut and stained with hematoxylin and eosin (HE). The
sections were analyzed, and images were captured using an
optical microscope (Nikon, Japan).
2.6 | Immunofluorescence staining
Testicular tissues and cells were fixed using 4% paraform-
aldehyde solution. For testicular tissues, the samples were
dehydrated in ethanol at different concentrations (70%, 90%,
and 100%). After embedding in paraffin blocks, the sections
were cut and then rehydrated (xylene, 5 minutes; ethanol,
100%, 95%, 70%, 5 minutes each). For immunostaining, the
sections and cells were permeabilized with 0.25% (v/v) Triton
X-100. After blocking with QuickBlock™ blocking buffer
(Beyotime), the slides were incubated with the primary anti-
bodies overnight at 4°C. The details of the antibodies used are
listed in Table S1. The next day, the samples were incubated
with secondary antibodies at room temperature in the dark for
2 hours, followed by 30 minutes incubation with 4′,6-diamid-
ino-2-phenylindole (DAPI). The stained slides were mounted
and observed using a fluorescence microscope (Nikon). The
fluorescence intensities were analyzed with Image-Pro Plus
6.0 software (Media Cybernetics).
2.7 | Western blot analysis
The treated cells were collected, and total protein was extracted
with RIPA lysis buffer containing 1 mmol/L phenylmethanesul-
fonyl fluoride. In some experiments, the nuclear proteins were
harvested using a Nuclear Protein Extraction Kit (Cat#: P0027;
Beyotime). Protein concentration was then measured using a
BCA protein assay kit (Beyotime). Equal amounts of protein
(20 μg) were subjected to electrophoretic separation using
a 10%-15% sodium dodecyl sulfate-polyacrylamide (SDS-
PAGE) gel and were subsequently transferred onto a nitrocellu-
lose membrane (Beyotime). After blocking with QuickBlock™
Blocking Buffer (Beyotime), membranes were incubated with
the primary antibodies overnight at 4°C. The details of the anti-
bodies used for Western blot assays are listed in Table S2. After
washing with TBST, the membranes were incubated with sec-
ondary antibodies at room temperature for 2 hours. The protein
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|    XU et al.
bands were exposed using a Bio-Rad imaging system (Bio-
Rad), and band intensities were quantified using the Quantity
One software (v. 4.52; Bio-Rad Laboratories).
2.8 | CCK-8 and Edu assays
Cell counting kit-8 (CCK-8, Beyotime) was used to exam-
ine cell viability. Briefly, the treated cells were seeded in
96-well plates and were incubated with 10 μL of CCK-8 so-
lution at 37°C for 1 hour. The optical density (OD) value
of each well at 450 nm wavelength was determined using
a microplate reader (BioTek). For 5-ethynyl-2-deoxyuridine
Edu assays, the proliferation ability of cells was evaluated
using a BeyoClick™ EdU Cell Proliferation Kit with Alexa
Fluor 594 (Cat#: C0078S; Beyotime). The procedure was
performed according to the manufacturer's instructions. After
Edu staining, cells were visualized using a fluorescence mi-
croscope (Nikon), and the percentage of Edu-positive cells
was calculated as the number of Edu-positive cells out of the
total number of cells (×100).
2.9 | Caspase3 activity and TUNEL assays
Caspase3 activity assays were performed using the Caspase3
Activity Assay Kit (Cat#: C1115; Beyotime). Briefly, treated
cells were lysed using lysis buffer. The supernatants of the
homogenate were harvested by centrifugation at 12 000 g,
and the protein concentration was measured using a BCA
protein assay kit. Subsequently, cell lysates were incubated
with Ac-DEVD-pNA (2 mmol/L) at 37°C for 2 hours. After
incubation, absorbance was read at 405 nm using a micro-
plate reader (BioTek).
For terminal deoxynucleotidyl transferase dUTP nick-end
labeling (TUNEL) assays, DNA damage analysis was per-
formed using a Colorimetric TUNEL Apoptosis Assay Kit
(Cat#: C1098; Beyotime). Testes and spermatogonia were
fixed in 4% (w/v) paraformaldehyde. After embedding in par-
affin blocks, the testicular sections (5 μm) were rehydrated and
washed in distilled water prior to TUNEL staining. Briefly,
the slides and spermatogonia were permeabilized with 0.3%
Triton X-100 for 10 minutes. Then, the endogenous peroxi-
dase of samples was inactivated with 0.3% H2O2 in PBS for
20 minutes. Samples were incubated with the TUNEL reac-
tion mixture at 37°C for 60 minutes and were stained with
streptavidin-HRP at room temperature for 30 minutes. The
nuclei were visualized using a hematoxylin staining solution.
Afterward, the samples were observed under a light micro-
scope (Nikon): TUNEL-positive cells were brown, and nu-
clei were stained as blue. The percentage of TUNEL-positive
cells was calculated as the number of brown cells out of the
total number of cells (×100).
2.10 | Measurement of lipid uptake
The fatty acid uptake assay was performed with Oil Red O so-
lution (Solarbio) and BODIPY lipid probes (Thermo Fisher).
For Oil Red O staining, cells were fixed in 4% formaldehyde
and were stained with Oil Red O solution for 30 minutes.
After washing, the nuclei were visualized with hematoxy-
lin staining solution and observed under a light microscope
(Nikon). Red indicates the lipid content. For BODIPY lipid
staining, testicular tissues were fixed in 4% formaldehyde,
and the frozen sections were permeabilized with 0.3% Triton
X-100. Slides and cells were incubated with BODIPY lipid
probes (10 μg/mL), and nuclei were stained with DAPI.
Immediately, the stained samples were observed using a
fluorescence microscope (Nikon). Fluorescence intensities
were analyzed using the Image-Pro Plus 6.0 software (Media
Cybernetics).
2.11 | Measurement of ROS and
MDA production
Intracellular ROS levels were observed with the fluorescent
dye 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA,
Beyotime). Briefly, the treated cells were incubated in
DMEM containing 10 µmol/L DCFH-DA for 30 minutes at
37°C. After washing, the labeled cells were observed using a
fluorescence microscope (Nikon). Malondialdehyde (MDA)
levels were evaluated using the Lipid Peroxidation MDA
Assay Kit (Cat#: S0131M; Beyotime) based on the manu-
facturer's instructions. Briefly, cells were lysed with RIPA
buffer, and protein concentration was determined using a
BCA kit. Cell lysates were incubated with test solution at
100°C for 15 minutes. Absorbance was then read at 532 nm
using a microplate reader (BioTek).
2.12 | Measurement of mitochondrial
distribution
For mitochondrial staining, treated cells were incubated
in DMEM containing 100 nmol/L Mito-Tracker Green
(Beyotime) in the dark at 37°C for 30 minutes. After wash-
ing, stained cells were observed using a fluorescence micro-
scope (Nikon). Fluorescence intensities were then analyzed
with Image-Pro Plus 6.0 software (Media Cybernetics).
2.13 | Flow cytometry assay
For the cell apoptosis assay, cells were evaluated using
an Annexin V-FITC Apoptosis Detection Kit (Cat#:
C1062M, Beyotime). After centrifugation, treated cells
XU et al.
were suspended in 500 μL of cold binging buffer and sub-
sequently incubated in this medium containing 5 μL of
Annexin V-FITC and 5 μL of PI, at room temperature for
10 minutes. The stained cells were detected using flow
cytometry (Beckman Co.) and analyzed using FlowJo
Software (FlowJo, LLC.).
For cell cycle distribution, cells were assessed using a Cell
Cycle Kit (Cat#: CA1510; Solarbio). Briefly, treated cells
were fixed in 70% ethanol at 4°C for 24 hours. After washing,
samples were stained with PI containing RNase A at 37°C
for 20 minutes and subsequently analyzed by flow cytometry.
Data were analyzed using ModFit software (Verity Software
House).
The mitochondrial membrane potential was evaluated
using a JC-1 Kit (Cat#: C2006, Beyotime). Treated cells
were incubated with JC-1 solution at 37°C for 20 minutes
and were detected using a flow cytometry 488-nm exci-
tation with 530-nm and 585-nm emission. Data were an-
alyzed using Flowjo software. The decrease in the red/
green fluorescence intensity ratio indicates mitochondrial
depolarization.
Intracellular Ca2+ levels were measured using Fluo-4 AM
(Beyotime). Treated cells were incubated with Fluo-4 AM
(2 µmol/L) solution at 37°C for 30 minutes and were eval-
uated by flow cytometry. Data were analyzed using FlowJo
Software.
ROS levels were determined by DCFH-DA staining.
Briefly, treated cells were incubated in DMEM containing
10 µmol/L DCFH-DA at 37°C for 30 minutes. After washing,
labeled cells were evaluated using flow cytometry. Data were
analyzed by FlowJo Software.
2.14 | Statistical analysis
All data are shown as the mean ± standard error of mean
(SEM). Statistical differences were analyzed by either
the two-tailed Student's t test or ANOVA followed by the
Student-Newman-Keuls test between two groups using SPSS
20.0 software (SPSS Inc). A P value <0.05 was considered
as statistically significant. All of these experiments were re-
peated at least three times unless specified otherwise.
3 | R E S U LTS
3.1 | Melatonin prevents testicular damage
caused by PA
To determine the effect of melatonin on the testes in re-
sponse to PA administration, we histopathologically ana-
lyzed the testes in the different treatment groups. As shown
in Figure 1A,B, after long-term PA injection (30 days), the
  
| 5 of 20
testicular mass in the PA-treated group was smaller than that
in the control group, and the testis/body ratio was also signif-
icantly reduced by PA treatment. Interestingly, a downward
trend of testicular mass caused by PA was ameliorated in mice
injected with melatonin (Figure 1A,B). Testicular hematoxy-
lin-eosin (HE) staining showed no obvious histopathological
abnormalities in the PA-treated group, whereas a larger hol-
low tendency was observed in some PA-treated seminifer-
ous tubules compared with the control group (Figure 1C). In
particular, PA induced a dramatic sperm loss, and melatonin
therapy significantly promoted spermatogenic recovery in
PA-injected mice (Figure 1C,D). As expected, epididymal
sperm concentration and motility deteriorated in the PA in-
jection group, which was ameliorated by pretreatment with
melatonin (Table 1). Sperm morphological analyses showed
that treatment with PA caused abnormal sperm morphology
and that this abnormal morphology was significantly relieved
when treatment was combined with melatonin (Figure 1E,F).
Furthermore, we mated PA- or PA/Mel-exposed male mice
to estrous females and evaluated the fertility of these males.
As shown in Figure 1G,H, the low pregnancy rate and re-
duced offspring caused by PA were significantly ameliorated
in the melatonin therapy group, suggesting that melatonin
can restore fertility in these PA-exposed male mice.
Consistent with the histopathological findings, the im-
munofluorescence assay demonstrated that the expression
of PLZF, a spermatogonial stem cell (SSC) specific marker
protein, was decreased after PA treatment for 30 days, which
was relieved by melatonin (Figure 1I,J). However, SOX9, a
marker of Sertoli cells, slightly decreased after PA treatment,
and pretreatment with melatonin had almost no significant
effect on the SOX9-positive cells in the testis (Figure S1).
The results suggested that the proliferation of SSCs was re-
covered by melatonin therapy. Moreover, endogenous plu-
ripotency genes, including Oct4 and SOX2, were examined
for their ability to self-renew testicular cells. These data
showed that both Oct4 and SOX2 were decreased by PA
treatment, which was relieved by pretreatment with mela-
tonin (Figure 1K,L,M), suggesting that melatonin restores
spermatogenesis in PA-treated mouse. To examine the cause
of the damaged testes, BODIPY lipid probes were used to
detect lipogenesis. As shown in Figure 1N, a dramatic in-
crease in fluorescence was observed in PA-treated testes,
suggesting lipid accumulation caused by PA. When treated
with melatonin, lipid accumulation was obviously attenuated
(Figure 1N). As excessive lipid accumulation in nonadipose
tissues can result in cellular lipotoxicity, upregulation of
TUNEL-positive cells was observed in the PA-treated sem-
iniferous tubules, which was relieved by melatonin treatment
(Figure 1O), indicating that melatonin can protect testicular
cells from PA-induced DNA damage. Taken together, these
results suggest that melatonin ameliorates the testicular dam-
age caused by PA.
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| 7 of 20

F I G U R E 1 Melatonin ameliorated testicular damage caused by PA. Male mice were intraperitoneally injected with PA (100 mg/kg/d)
with or without melatonin (10 mg/kg/d) for 30 d. (A) Morphology of testes in the different groups. (B) The ratio of teste/weight in the different
groups. (C) Representative images of testicular cross sections with HE staining in the different groups. Scale bars = 100 μm. (D) The number of
spermatozoa in the tubule cross sections from different treatment groups. (E) Sperm morphology in the control (n = 8), PA (n = 6), PA + Mel
(n = 6)-treated groups. (F) The percentage of normal sperm per epididymis. (G) Pregnancy rate in the different groups. (H) The number of
offspring in the different treatment males. (I, K) Representative immunofluorescent images show the expression of PLZF, Oct4, and SOX2 in the
different groups. Scale bars = 200 μm. (J, L, M) The percentage of PLZF-positive, Oct4-positive and SOX2-positive cells was shown, respectively.
(N) Representative images of BODIPY staining in the different groups. Scale bars = 200 μm. Quantification of the BODIPY fluorescence
intensity was shown. (O) Representative images of TUNEL staining in the different groups. Brown corresponds to TUNEL-positive cells, whereas
blue corresponds to nuclei. Scale bars = 300 μm. Percentage of TUNEL-positive cells was shown. Data are representative of three independent
experiments with similar results. *P < .05; **P < .01; ***P < .001

3.2 | Melatonin suppresses PA-
induced apoptosis
To further confirm which cell type in the testis was more
vulnerable to PA, cell line viability was assessed with the
CCK-8 assay at gradient concentrations of PA in vitro. As
shown in Figure 2A, cell viability for type B SSCs (GC1)
and type A SSCs (C18-4) was more vulnerable than that of
the Leydig cells (MLTC1). In particular, the cytotoxic ef-
fect of PA appeared to dramatically increase at concentra-
tions higher than 100 μmol/L in GC-1 cells (Figure 2A).
Interestingly, pretreatment with melatonin at concentrations
of 0.1, 1.0, and 10.0 μmol/L significantly ameliorated the
cell viability of GC-1 cells exposed to 100 μmol/L PA for
24 hours, and melatonin at a concentration of 1.0 μmol/L was
shown to be most beneficial in terms of conferring resistance
to cytotoxicity (Figure 2B). Following this, a concentration
of 100 μmol/L PA and 1.0 μmol/L melatonin was used in
subsequent experiments unless otherwise stated.
To ascertain whether PA-induced cytotoxicity occurs
through the apoptotic pathway, a flow cytometry assay
was carried out. As shown in Figure 2C, a significant in-
crease in apoptotic cells was observed in PA-exposed cells.
Compared with the control group, treatment with melatonin
alone did not alter the rate of apoptotic cells, whereas pre-
treatment with melatonin significantly alleviated PA-induced
apoptosis (Figure 2C). To confirm these results, the activ-
ity of Caspase3, a critical player in apoptosis, was detected
using a Caspase3 activity assay kit. As shown in Figure 2D,
treatment with PA significantly increased Caspase3 activ-
ity, while melatonin therapy effectively relieved this PA-
induced upregulation. Western blot analysis also showed a
marked increase in the protein expression of cleaved forms
of Caspase3 in the PA-treated group, while the BCL2/Bax
ratio was decreased (Figure 2E–G). Similarly, protein ex-
pression levels of pro-apoptotic proteins were suppressed by
pretreatment with melatonin (Figure 2E–G). Given that endo-
plasmic reticulum stress (ERS) is one of the main causes of
apoptosis, an increase of ERS-related apoptotic markers, in-
cluding Cleaved-Caspase12 and CHOP, was observed in PA-
treated cells, which was then relieved by melatonin therapy
(Figure 2E,H,I). These findings suggest that melatonin inhib-
its PA-induced cell apoptosis due to downregulation of apop-
totic proteins.
3.3 | Melatonin relieves PA-induced
ER stress
Because of the changes in the expression of ERS-related ap-
optotic proteins, protein levels of ER stress markers were de-
termined. PA treatment alone clearly increased p-IRE1 and
p-PERK signals as well as ATF4 expression, suggesting that
drastic ER stress occurred in PA-exposed cells (Figure 3A-
D); however, sodium phenylbutyrate (4-PBA), an ER stress
inhibitor, significantly decreased the phosphorylation levels
of IRE1 and PERK, and ATF4 expression in PA-treated cells
(Figure 3A-D). Consistent with 4-PBA’ action, these changes
in ER stress markers can be alleviated with the efficacy of
melatonin therapy (Figure 3A-D). ER stress could disturb
calcium homeostasis, which affects proper protein folding
and transport. Functionally, intracellular Ca2+ levels were de-
tected with Fluo-4 AM staining, and flow cytometry showed
that a dramatic increase in intracellular Ca2+ concentration
was observed in PA-treated cells, suggesting disturbed ER
functions caused by PA (Figure 3E). Furthermore, impaired
ER function caused by PA treatment partially recovered
when the cells were co-treated with melatonin or 4-PBA
(Figure 3E). These results indicate that melatonin effectively
blocks PA-induced ER stress and maintains ER homeostasis.
3.4 | Melatonin ameliorates PA-induced
lipotoxicity by attenuating oxidative stress
To further reveal the potential involvement of melatonin
in the protective mechanism of the testes against PA-
induced lipotoxicity, we next measured Nrf2 signals in the
PA-injected mice, which were thought to be involved in
resistance to oxidative stress. As shown in Figure 4A, phos-
phorylation of Nrf2 (p-Nrf2) was increased in PA-treated
testes, which was partially relieved by melatonin treatment.
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Parameters Control (n = 8)
6 a
Concentration (10 /mL) 16.03 ± 1.37
Motility (%) 71.28 ± 2.17a
Note: Data are represented as the mean percentage ± SEM. Values within a row with different letters (a, b, c)
indicate significant differences (P < .05).
Oil Red O staining and BODIPY lipid probes showed that
PA treatment resulted in increased lipid accumulation
(Figure 4B); however, pretreatment with melatonin barely
inhibited fatty acid uptake in vitro (Figure 4B), which was
a result different from that of the testes in vivo. As cyto-
toxic accumulation of lipids in nonadipose tissues might
lead to cellular lipotoxicity, a significant increase in malon-
dialdehyde (MDA) levels was observed in PA-treated sper-
matogonia, which was considered to be the key mechanism
behind ROS-induced sperm damage (Figure 4C); however,
melatonin pretreatment prevented the MDA production by
lipid peroxidation (Figure 4C). To determine whether PA
is involved in oxidative stress, a DCFH-DA probe and flow
cytometry analysis were used to monitor cellular ROS. As
shown in Figure 4D,E, PA treatment drastically increased
intracellular ROS accumulation, and melatonin relieved
elevated ROS generation via its efficient antioxidant ac-
tivity. However, there was no significant change observed
in the ROS content when comparing melatonin alone and
the control groups (Figure 4D,E). Furthermore, stress-re-
lated extracellular signal-regulated kinase (ERK) and p38
MAPK signaling pathways were investigated using Western
blot analysis. The data showed that no significant change
in ERK1/2 signals was observed in the different treatment
groups, while a significant activation of p38 MAPK sign-
aling was observed in the PA-treated cells (Figure 4F-H).
Similarly, the p-Nrf2 levels were increased by PA admin-
istration (Figure 4F,I). Consistent with the ROS generation
pattern, melatonin alone did not significantly change the ex-
pression levels of p-p38 MAPK and p-Nrf2 (Figure 4F,H,I).
However, PA-induced stimulation was relieved by mela-
tonin therapy (4F, H, I). Given that phosphorylated Nrf2 has
been considered to drive its subcellular nuclear localization,
we set out to evaluate the correlation between melatonin
and Nrf2 signals. As expected, immunofluorescence re-
sults showed that an increase in the amount of nuclear Nrf2
was observed in PA-exposed cells (Figure 4J), suggesting
that PA-induced ROS is accompanied by Nrf2 activation.
Interestingly, BMS-582949, a p38 MAPK antagonist, abol-
ished PA-induced nuclear accumulation of Nrf2, and mela-
tonin partially mimics this action (Figure 4J). Based on these
findings, it is indicated that Nrf2 activation caused by p38
MAPK signaling is compensatory for the response to PA-
induced oxidative stress, but it is not enough to reduce ROS
T A B L E 1 Melatonin relieved
PA + Mel
the deterioration of epididymal sperm
PA (n = 6) (n = 6)
parameters caused by PA
8.89 ± 1.24c 12.96 ± 1.54b
42.74 ± 1.92c 64.36 ± 2.26b
levels, and that melatonin therapy can relieve this stress via
actions that are yet-unidentified.
3.5 | Melatonin stimulates SIRT1 pathway
through melatonin receptors in response to PA-
induced damage
We then further examined the melatonin-mediated signaling
pathways. As shown in Figure 5A, melatonin receptors (MT1
and MT2) were observed in SSCs, Leydig cells, and Sertoli
cells of the mouse testes; however, long-term PA injection
alone did not significantly change the expression of mela-
tonin receptors. Compared with the control and PA-treated
groups, higher fluorescent MT1 and MT2 signals were ob-
served in testes co-treated with melatonin (Figure 5A). To
investigate whether melatonin also promoted melatonin re-
ceptor expression in vitro, spermatogonial stem cell lines
(GC-1) were exposed to various concentrations of melatonin
(0.1, 1.0, and 10.0 μmol/L). As expected, melatonin treatment
significantly enhanced MT1 and MT2 expression levels, and
that the most stimulatory effect of melatonin was found at
1.0 μmol/L concentration (Figure 5B). To investigate whether
sirtuins are involved in the response of spermatogonia to me-
latonin treatment, protein levels of SIRT1, SIRT2, and SIRT3
were measured. Western blot analysis showed that expres-
sion levels of SIRT1, SIRT2, and SIRT3 in PA-treated cells
were significantly lower than those in the control group; how-
ever, the downregulation effect of PA treatment on SIRT2
and SIRT3 expression was not recovered through melatonin
therapy (Figure 5C). Interestingly, melatonin administration
abolished the suppressive effect of SIRT1 expression in PA-
treated cells (Figure 5C). Based on these results, we deter-
mined whether the melatonin-regulated increase in SIRT1
occurred through its receptors. The spermatogonia were
pretreated with luzindole, a melatonin receptor antagonist,
prior to melatonin administration. As shown in Figure 5D,
the stimulatory effect of melatonin on SIRT1 expression
was abolished by luzindole treatment of PA-exposed cells.
Furthermore, immunofluorescence analysis showed that MT1
fluorescent signals were positively correlated with SIRT1
during the co-treatment of PA with melatonin (Figure 5E,F).
Conversely, this relationship between melatonin and SIRT1
was abrogated by luzindole pretreatment (Figure 5E,F).
XU et al.   
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F I G U R E 2 Melatonin relieved PA-induced apoptosis. (A) The viability of cell line was analyzed at gradient concentrations of PA treatment
for 24 h in type A SSCs (C18-4), type B SSCs (GC-1), and Leydig cells (MLTC1). (B) Effect of pretreatment with melatonin on the viability of PA
(100 μmol/L)-exposed GC-1 cells at different times. (C) Spermatogonia were pretreated with melatonin (1.0 μmol/L) for 2 h and PA (100 μmol/L)
was added for an additional 24 h. Apoptosis of spermatogonia was detected by flow cytometry analysis in the control, Mel, PA, PA + Mel-treated
groups. Percentages of the apoptotic cells were shown. (D) Caspase3 activity was shown in the different groups. (E) Western blot analysis of BCL2,
Bax, Cleaved-Caspase3, Cleaved-Caspase12, CHOP in the different groups. (F-I) The bar graph showed the quantification of Bax/BCL2, Cleaved-
Caspase3/Tubulin, Cleaved-Caspase12/Tubulin, and CHOP/Tubulin, respectively. Data are representative of three independent experiments with
similar results. *P < .05; **P < .01

Subsequently, quantitative immunofluorescence revealed a 3.6 | Melatonin relieves PA-induced

decrease of over 4-fold in the subcellular nuclear localization
of SIRT1 in PA-treated cells, which was restored by melatonin
administration (Figure 5E,G). However, melatonin-enhanced
subcellular nuclear localization of SIRT1 was abolished by
luzindole treatment (Figure 5E,G). Collectively, these find-
ings suggest that melatonin may rely on its receptors to acti-
vate SIRT1 pathway.
oxidative stress through the MT/SIRT1/FoxO1
signaling pathways
To investigate how melatonin protects against PA-induced
oxidative stress, forkhead box protein O1 (FoxO1), a tran-
scription factor that acts as a regulator of cell response to
oxidative stress, was analyzed in spermatogonia (GC-1). As
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F I G U R E 3 Melatonin relieved
PA-induced endoplasmic reticulum stress
(ERS). The spermatogonia were cultured
with melatonin (1.0 μmol/L) for 2 h with or
without ERS inhibitor 4-PBA (250 μmol/L),
prior to PA treatment (100 μmol/L) for 24 h.
Cells without any treatment were used as
blank controls. (A) Western blot detection
of ERS-related proteins p-IRE1, IRE1,
p-PERK, PERK, and ATF4 in the control,
PA, PA + Mel, and PA + 4-PBA treated
groups. (B-D) The bar graph showed the
quantification of p-IRE1/IRE1, p-PERK/
PERK, and ATF4/Tubulin, respectively.
(E) Flow cytometry analysis showed
intracellular Ca2+ levels in the different
groups. Data are representative of three
independent experiments with similar
results. *P < .05; **P < .01; ***P < .001

shown in Figure 6A, a significant increase in FoxO1 acety-
lation was detected in PA-treated cells, which was recov-
ered by melatonin therapy. Interestingly, co-incubation of
spermatogonia with either an MT antagonist (luzindole) or
a SIRT1 antagonist (EX527) abolished the downregulation
effect of melatonin on acetylated FoxO1 (Figure 6A), sug-
gesting that melatonin inhibits FoxO1 acetylation via the
activation of MT/SIRT1 signals in PA-stimulated cells. To
determine whether melatonin could modulate downstream
SIRT1 signaling pathway, the protein expression levels of
antioxidant enzymes, including Catalase and MnSOD, were
investigated. Consistent with PA-induced oxidative stress,
PA treatment resulted in low levels of Catalase and MnSOD
(Figure 6A), indicating that the cellular antioxidant system

F I G U R E 4 Melatonin ameliorated PA-induced lipotoxicity by attenuating oxidative stress. (A) Representative immunofluorescent images
showed p-Nrf2 expression in mouse testes depending on the different treatments. Scale bars = 200 μm. The fluorescence intensity of p-Nrf2
was shown from 12 testicular tissues. (B) Representative images of lipid content in spermatogonia by Oil Red O (Scale bars = 100 μm.) or
BODIPY (Scale bars = 200 μm.) staining. Quantification of the BODIPY fluorescence intensity was shown in the different groups. (C) Levels
of malondialdehyde (MDA) showed lipid peroxidation in the different groups. (D) Representative images of cellular ROS levels in the different
groups. Scale bars = 200 μm. (E) Flow cytometry analysis showed the intracellular ROS content in the different groups. (F) Protein expression
levels of p-ERK, ERK, p-p38 MAPK, p38 MAPK, and p-Nrf2 in the different groups. (G-I) The bar graph showed quantification of p-ERK/ERK,
p-p38 MAPK/p38 MAPK, and p-Nrf2/Tubulin, respectively. (J) Immunofluorescence staining showing the subcellular localization of the Nrf2
protein (red) and the nuclei stain DAPI (blue) in spermatogonia. Scale bars = 100 μm. Quantification of the colocalization efficiency between Nrf2
and DAPI in the different groups. Data are representative of three independent experiments with similar results. *P < .05; **P < .01; ***P < .001
XU et al.   
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is damaged at a high level of ROS. However, a significant by either luzindole or EX527. These results confirmed that
upregulation of Catalase and MnSOD was observed after co- melatonin promoted SIRT1-dependent antioxidant signal-
treatment of PA with melatonin, which was then abrogated ing. Furthermore, Western blot analysis of spermatogonia
XU et al.   
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F I G U R E 5 Melatonin activated SIRT1 pathway through melatonin receptors in response to PA-induced damage. (A) Representative
immunofluorescent images showed the expression of MT1 (green) and MT2 (red) in mouse testes with different treatment. Fluorescence intensity
of MT1 and MT2 was shown from 12 testicular tissues, respectively. Scale bars = 200 μm. (B) The effect of melatonin on MT1 and MT2 at
different concentrations (0.1, 1.0, 10 μmol/L). Fluorescence intensity of MT1 and MT2 was shown in spermatogonia. Scale bars = 200 μm. (C)
Protein expression levels of SIRT1, SIRT2, SIRT3 in the control, PA (100 μmol/L), and PA (100 μmol/L) + Mel (1.0 μmol/L) treated groups.
The bar graph showed the quantification of SIRT1, SIRT2, and SIRT3, respectively. (D) Western blot detection of SIRT1 in the control, Mel
(1.0 μmol/L), PA (100 μmol/L), PA (100 μmol/L) + Mel (1.0 μmol/L), PA (100 μmol/L) +Mel (1.0 μmol/L) + Luz (10 μmol/L)-treated groups. (E)
Immunofluorescence staining showing the levels of MT1 (green) and SIRT1 (Red), and the subcellular localization of the SIRT1 in spermatogonia.
The arrowhead showed colocalization efficiency between SIRT1 and the nucleus. (F) Fluorescence intensities of SIRT1 were shown. (G)
Quantification of the colocalization efficiency between SIRT1 and DAPI in the different groups. Data are representative of three independent
experiments with similar results. *P < .05; **P < .01; ***P < .001

nuclear extracts to determine the subcellular localization of
FoxO1 was performed. Consistently, compared to the control
group, PA administration significantly inhibited the subcel-
lular nuclear localization of FoxO1, whereas treatment with
melatonin mostly restored nuclear FoxO1 accumulation in
PA-exposed cells (Figure 6B). Meanwhile, melatonin-in-
duced nuclear accumulation of FoxO1 was completely abol-
ished by either luzindole or EX527. Strikingly, a negative
correlation was observed between FoxO1 acetylation and
nuclear localization due to melatonin. This indicates that me-
latonin enhances FoxO1 activation via a SIRT1-dependent
deacetylation activity, thereby affecting downstream targets.
Similar findings were obtained, wherein the ameliorative ef-
fect of melatonin on PA-induced oxidative stress was mostly
abolished by either the MT antagonist or SIRT1 antagonist
(Figure 6C). Taken together, these findings suggest that me-
latonin protects spermatogonia against PA-induced oxidative
stress through the activation of MT/SIRT1/FoxO1 signaling
pathways.
Conversely, PA-induced mitochondrial impairment was
partially recovered by melatonin pretreatment, which was
then abolished by treatment with either luzindole or EX527
(Figure 7C,D). This suggests that downregulating MT/SIRT1
pathways with inhibitors prevents the protective effect of
melatonin against PA-induced mitochondrial damage. Given
that PGC-1α is a key transcription factor that stimulates mito-
chondrial biogenesis, the protein expression of PGC-1α was
then detected. PA treatment decreased PGC-1α expression,
whereas retreatment with melatonin helped in the recovery
of PGC-1α expression (Figure 7E,F). Meanwhile, treatment
with either luzindole or EX527 prevented the upregulation of
PGC-1α mediated by melatonin (Figure 7E,F). These find-
ings suggest that melatonin prevents mitochondrial dysfunc-
tion caused by PA in a SIRT1-dependent manner.
3.8 | Melatonin recovers spermatogonia
proliferation in response to DNA damage
caused by PA through p53 deacetylation

3.7 | Melatonin prevents PA-induced
mitochondrial dysfunction in a SIRT1-
dependent manner
Given that a high level of ROS may directly contribute to
mitochondrial dysfunction, which in turn decreases anti-
oxidant enzymes and exacerbates oxidative stress, we next
evaluated the role of melatonin on mitochondrial function
in PA-induced spermatogonia. As expected, flow cytom-
etry analysis by JC-1 staining revealed a lower measurement
of mitochondrial membrane potential in PA-treated cells
(Figure 7A,B), suggesting that PA impairs mitochondrial
function. This effect was attenuated by melatonin therapy.
However, the protective effects of melatonin on mitochon-
drial functional capacity were partially abolished by either
luzindole or EX527, indicating that melatonin maintains
mitochondrial function via MT/SIRT1 signaling pathways.
Furthermore, we detected the mitochondrial quantity using
Mito-Tracker Green. As shown in Figure 7C,D, PA-exposed
cells showed decreased mitochondrial mass, as seen in the
decline of the Mito-Tracker Green fluorescence signals.
We further explored how melatonin alleviates the loss
of testicular SSCs caused by PA. As shown in Figure 8A,
PA treatment increased DNA damage, as measured by the
TUNEL assay. However, co-treatment with melatonin sig-
nificantly reduced PA-induced DNA damage, as indicated
by the decline of TUNEL-positive cells; this protective ef-
fect was abolished by the SIRT1 inhibitor (Figure 8A).
Consistent with this observation, flow cytometry analysis
revealed that PA treatment drastically increased the number
of cells in the G2/M phase compared with the control cells,
suggesting that DNA damage caused by PA leads to cell
cycle arrest. However, cell cycle arrest in the G2/M phase
was prevented when the spermatogonia were co-treated with
melatonin (Figure 8B). In addition, melatonin-induced cell
division was abrogated by the SIRT1 inhibitor (Figure 8B).
In line with this, a significant inhibition of spermatogonia
proliferation was observed in PA-treated cells as observed
through Edu staining, which was then recovered by pretreat-
ment with melatonin (Figure 8C). Moreover, the SIRT1
inhibitor reversed the stimulatory effects of melatonin on
cell proliferation (Figure 8C). To confirm these results, the
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F I G U R E 6 Melatonin relieved PA-induced oxidative stress through MT/SIRT1/FoxO1 signaling pathways. Spermatogonia were cultured
with melatonin (1.0 μmol/L) for 2 h with or without the melatonin receptor inhibitor luzindole (10 μmol/L) or SIRT1 inhibitor EX527 (2.0 μmol/L)
before treatment with PA (100 μmol/L) for 24 h. Cells without any treatment were used as blank controls. (A) Western blot analysis showed
the protein levels of Ac-FoxO1, FoxO1, Catalase, and MnSOD in the control, PA, PA + Mel, PA + Mel +Luz, and PA + Mel +EX527-treated
groups. The bar graph showed quantification of Ac-FoxO1/FoxO1, Catalase/Tubulin, MnSOD/Tubulin, respectively. (B) Western blots of nuclear
FoxO1 protein in the different groups. The bar graph showed relative quantification of nuclear FoxO1 protein normalized to Histone H3. (C) Flow
cytometry analysis showed the intracellular ROS content in the different groups. Data are representative of three independent experiments with
similar results. Bars with different letters (a, b, and c) indicate significant differences

proliferative protein PCNA was detected by an immuno-
fluorescence assay. Results showed that PA treatment mark-
edly reduced the number of PCNA-positive cells, while
pretreatment with melatonin increased PCNA-positive cells
(Figure 8D). Interestingly, the melatonin-induced increase in
PCNA-positive cells was suppressed by the SIRT1 inhibitor
(Figure 8D). Given the above results, we further measured
the levels of acetylated p53, which was involved in cell cycle
regulation in response to DNA damage. Western blot analy-
sis showed that PA treatment drastically induced acetylated
p53 at a high level, whereas pretreatment with melatonin
recovered the normal levels of acetylated p53. Consistent
with SIRT1-mediated DNA damage in spermatogonia, the
downregulation effect of melatonin on acetylated p53 was
abolished by the inhibition of SIRT1 activity with EX527
(Figure 8E). Overall, the experimental results suggest that
melatonin depends on SIRT1-mediated p53 deacetylation,
which relieves G2/M arrest in response to PA-stimulated
DNA damage.
4 | D IS C U SS ION
In nonadipose tissue, excessive uptake of fatty acids such as
palmitic acid (PA) leads to elevated generation of deleterious
lipids, impaired cellular function, and oxidative stress-related
disease.4 In particular, oxidative stress is a major cause of
male infertility because testicular tissue has a very high rate
of cell division and mitochondrial oxygen consumption as
well as higher levels of fatty acids compared to other tis-
sues.35 Melatonin is a well-known powerful antioxidant and
is an endogenous hormone involved in multiple cellular pro-
cesses such as metastasis,36 proliferation,37 and apoptosis.38
However, it is still unclear whether melatonin attenuates PA-
induced damage in testicular cells. Here, we provided direct
evidence, both in vivo and in vitro, that melatonin protects
against lipotoxicity caused by PA through the attenuation of
oxidative stress. Furthermore, this study demonstrated that
the SIRT1 signaling pathway properties of melatonin are re-
sponsible for the alleviation of PA-induced lipotoxicity.
In this study, long-term PA injection in vivo led to lipid
accumulation and testicular injury, especially spermatogo-
nial loss. Numerous studies have indicated that melatonin
protects testicular cells against adverse conditions such as
hyperthermia, environmental toxins, and cytotoxic drugs.18-
20
Similarly, melatonin alleviated PA-induced testicular dam-
age and restored fertility in a mouse model. To elucidate the
mechanism underlying the beneficial effect of melatonin
in response to PA-induced lipotoxicity, type B SSCs were
utilized. ER stress is one of the main causes of apoptosis.
According to our in vitro data, melatonin inhibited the ex-
pression of pro-apoptotic proteins, particularly caspase12 and
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F I G U R E 7 Melatonin prevented PA-induced mitochondrial dysfunction in a SIRT1-dependent manner. Spermatogonia were cultured with
melatonin (1.0 μmol/L) for 2 h with or without melatonin receptor inhibitor luzindole (10 μmol/L) or SIRT1 inhibitor EX527 (2.0 μmol/L),
before treatment with PA (100 μmol/L) for 24 h. Cells without any treatment were used as blank controls. (A) Flow cytometry analysis showed
mitochondrial membrane potential via JC-1 staining. (B) The ratios of red/green fluorescence intensity were shown. (C) Representative images of
mitochondrial distribution in the different groups. Scale bars = 200 μm. (D) Fluorescence intensity of Mito-Tracker Green staining was shown.
(E) Western blot analysis showed PGC-1α expression in the different groups. (F) The bar graph showed the relative quantification of PGC-1α
normalized to Tubulin. Data are representative of three independent experiments with similar results. Bars with different letters (a, b, c, d) indicate
significant differences
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F I G U R E 8 Melatonin recovered proliferation in response to DNA damage caused by PA through p53 deacetylation. Spermatogonia were
treated with melatonin (1.0 μmol/L) for 2 h with or without SIRT1 inhibitor EX527 (2.0 μmol/L), before treatment with PA (100 μmol/L) for 24 h.
Cells without any treatment were used as blank controls. (A) Representative images of TUNEL staining in spermatogonia with different treatments.
Dark brown corresponds to TUNEL-positive cells, whereas blue corresponds to nuclei. Scale bars = 200 μm. The bar graph showed the percentage
of TUNEL-positive cells in the different groups. (B) Cell cycle distribution was shown by flow cytometry in the different groups. The bar graph
showed the percentage of spermatogonia in G0/G1, S, G2/M phase. (C) Representative images of Edu staining in spermatogonia under different
treatments. Scale bars = 200 μm. The bar graph showed the percentage of Edu-positive cell. (D) Representative immunofluorescent images of
PCNA expression in the different groups. Scale bars = 100 μm. The bar graph showed the percentage of PCNA-positive cells. (E) Western blot
analysis showed Ac-p53 expression in the different groups. The bar graph showed the relative quantification of Ac-p53 normalized to Tubulin.
Data are representative of three independent experiments with similar results. **P < .01; ***P < .001

CHOP, which are associated with ER stress. Subsequently,
changes in ER stress markers showed that melatonin relieved
PA-induced ER stress. This is in agreement with previous
studies confirming that melatonin can relieve spermatogonial
stem cell apoptosis by alleviating ER stress.39,40 Indeed, mel-
atonin directly and indirectly ameliorates ER stress in mul-
tiple pathologies. Melatonin directly promotes extracellular
secretion of insulin in INS-1E cells under induced ER stress
conditions.41 Melatonin also functions as an anti-inflamma-
tory therapy via ER stress in acute pancreatitis.42 Based on
previous studies and our results, melatonin may repair the ad-
verse effects of PA on male infertility by inhibiting ER stress.
Growing evidence has revealed that ER stress and oxida-
tive stress as closely linked events play crucial roles in cel-
lular apoptosis. Published data have suggested that excessive
PA stimulates oxidative stress in multiple cell lines such as
HUVECs and podocytes.43,44 Likewise, Shi et al45 reported
that PA-stimulated ROS generation is accompanied by acti-
vation of Nrf2 pathway. In this study, PA induced phosphor-
ylated Nrf2 at a high level by activating p38 MAPK pathway,
which is involved in the resistance to oxidative stress in PA-
injected mice. Interestingly, melatonin therapy did not acti-
vate Nrf2, but instead downregulated it by inhibiting the p38
MAPK pathways in PA-exposed spermatogonia in the pres-
ent study. In contrast, Wang et al46 reported that melatonin
activates the Nrf2 pathway in response to brain injury. These
raise the possibility that melatonin may alleviate the cellular
oxidative stress response by directly reducing ROS produc-
tion in spermatogonia. Subsequent experiments confirmed
that melatonin exerted beneficial effects in response to PA-
induced oxidative stress. Sirtuins are emerging as mediators
of cellular response in various oxidative stress-mediated
pathological situations.47 Here, the two major subtypes of
melatonin receptors, MT1 and MT2, were present in testicu-
lar cells. Furthermore, expression and activity of SIRT1 were
stimulated by melatonin therapy in response to PA admin-
istration through its receptors, but SIRT2 and SIRT3 were
not. Similar to our observations, previous reports have also
shown that melatonin increased SIRT1 expression in mouse
testicular cells,20,48 indicating a possible functional interac-
tion between melatonin and SIRT1 in PA-induced oxidative
stress. Hence, we used special antagonists to investigate the
anti-stress effects of melatonin and its underlying mecha-
nism, and we focused on the potential involvement of SIRT1
in its regulation. Interestingly, our results show that the pro-
tective effects of melatonin on PA-induced oxidative stress
were abolished by SIRT1 inactivation. Moreover, previous
studies have found that FOXO transcription factors contrib-
ute to cellular oxidative stress via the SIRT1 deacetylase.49,50
Our observations are consistent with those of previous’ stud-
ies showing that melatonin-induced SIRT1 activation inhib-
ited acetylated FoxO1 drove its nuclear localization, thereby
synthesizing antioxidant enzymes including Catalase and
MnSOD. Studies have shown that SIRT1 deacetylates ly-
sine residues in the FoxO1 DNA-binding domain and pro-
motes nuclear retention of FoxO1, thereby upregulating
its transcriptional activity.51,52 These findings suggest that
melatonin relieves PA-induced oxidative stress through the
SIRT1/FoxO1 signaling pathways.
Mitochondrial dysfunction has been considered as a major
factor inducing cell death,53 specifically in testicular tissue
with high mitochondrial oxygen consumption. Strong evidence
indicates that palmitate induces cell apoptosis through the ac-
tivation of the mitochondria-mediated apoptosis pathway.54
Consistently, we found that PA activated the mitochondrial
apoptosis pathway, including Bcl-2 inhibition and Bax promo-
tion, as well as Caspase3 activation. Furthermore, the beneficial
effects of melatonin on mitochondrial function inhibited mito-
chondrial oxidative apoptosis in the presence of PA. Indeed,
melatonin has been reported to prevent mitochondrial dysfunc-
tion in response to oxidative stress.55 Here, we showed that
melatonin prevented mitochondrial respiration, as indicated by
the upregulation of MMP and mitochondrial biogenesis. In ad-
dition, melatonin recovered the expression of PGC-1α, which
regulates mitochondrial biogenesis and mitochondrial fatty
acid oxidation, as a substrate for SIRT1. Similar to our findings,
Gerhart-Hines et al56 demonstrated that SIRT1/PGC-1α path-
ways regulate mitochondrial function and fatty acid oxidation in
muscle. However, the ameliorative effects of melatonin on mi-
tochondrial dysfunction caused by PA were abolished by either
an MT antagonist or a SIRT1 antagonist, indicating that mel-
atonin protects against PA-induced mitochondrial dysfunction
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F I G U R E 9 A possible mechanism by which melatonin protected
spermatogonia from palmitic acid-induced lipotoxicity. Melatonin
prevented PA-induced oxidative stress through MT/SIRT1/FoxO1
signaling pathways and subsequently alleviated mitochondrial
dysfunction and DNA damage caused by redox imbalance via the
modulation of PGC1α and P53 in a SIRT1-dependent manner, thereby
inhibiting apoptotic cell death in spermatogonia. PA, palmitic acid;
MT, melatonin receptor; ΔΨm, mitochondrial membrane potential
and maintains mitochondrial function by upregulating PGC-1α
in a SIRT1-dependent manner. These data are in accordance
with literature demonstrating the melatonin-mediated ameliora-
tion of mitochondrial dysfunction through SIRT1 activation.57
Studies have shown that DNA damage is largely due to
oxidative stress which induces defective spermatozoa func-
tion.58,59 Cells have evolved mechanisms to retard prolifera-
tion in response to DNA damage.60 In the present study, we
observed that PA-induced oxidative stress resulted in DNA
damage, leading to G2/M arrest and subsequent cell death in
spermatogonia. Melatonin has been shown to prevent DNA
damage caused by oxidative stress in several different tissue
cell types including liver61 and brain.62 Consistent with pre-
vious reports, we found that melatonin prevented DNA dam-
age and promoted the proliferation of testes in response to
PA stimulation. It is worth noting that these beneficial effects
were abolished by SIRT1 inactivation. Further experiments
revealed that PA-induced DNA damage caused acetylated
p53 accumulation, whereas melatonin decreased this accu-
mulation via the deacetylase activity of SIRT1. Indeed, p53 is
a deacetylated substrate for SIRT1,63 which in turn becomes
acetylated in response to DNA damage, and the acetylated
form increased its transcriptional activity, thereby leading to
cell cycle arrest.64,65 These findings are consistent with the
notion that melatonin protects spermatogonia against PA-
induced DNA damage and subsequently inhibits G2/M arrest
through the SIRT1/p53 signaling pathways.
In conclusion, this study demonstrated that melatonin
protected spermatogonia against PA-induced lipotoxicity
through a complex mechanism (Figure 9). Melatonin pre-
vents the PA-induced oxidative stress, ER stress, mitochon-
drial dysfunction, and DNA damage via the modulation of
FoxO1, PGC1α, and P53 in a SIRT1-dependent manner,
thereby alleviating lipotoxicity caused by PA. These findings
suggest that melatonin may be an attractive agent for attenu-
ating obesity-associated male infertility.
ACKNOWLEDGEMENTS
This work was supported by the Ministry of Agriculture
Transgenic Major Projects (2018ZX0801013B),
Key Industry Innovation Chain of Shaanxi Province
(2018ZDCXL-NY-02-06), and National Key Technology
Support Program (2015BAD03B04).
CONFLICT OF INTEREST
The authors declare no competing financial interest.
AUTHOR CONTRIBUTION
Q. L. provided financial support and conceived the re-
search study. D. X. performed the experiments, wrote the
manuscript. L. L. revised the manuscript. Y. Z. edited the
manuscript. L. Y., J. C. and R. H. conducted the animal ex-
perimentation, histological analysis and biochemical analy-
sis. Z. Z. analyzed the data. All authors read and approved
the final manuscript.
ORCID
Qingwang Li https://orcid.org/0000-0001-7638-1565
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SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Xu D, Liu L, Zhao Y, et al.
Melatonin protects mouse testes from palmitic acid-
induced lipotoxicity by attenuating oxidative stress and
DNA damage in a SIRT1-dependent manner. J Pineal
Res. 2020;69:e12690. https://doi.org/10.1111/jpi.12690