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Background: The impact of melatonin on bisphenol A (BPA)-induced
testicular apoptosis and endoplasmic reticulum (ER) stress was explored.
Methods: The mice received BPA (50 mg/kg) by gavage for 30 days while
being injected with 20 mg/kg melatonin. Protein expressions were detected with
western blotting. The Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labeling (TUNEL) assay measured testicular cell apoptosis. Testosterone was
quantified by employing enzyme-linked immunosorbent assay (ELISA).
Results: Melatonin promoted the development of seminiferous tubules,
restored the orderly arrangement of the germ cells, and increased epithelial
layers in the seminiferous tubules in BPA-treated mice. Moreover, in BPA-treated
mouse testicular cells, melatonin markedly upregulated melatonin receptor 1A
(MTNR1A) and melatonin Receptor 2 (MTNR2) expressions while downregulating ER
molecular chaperones glucose-regulated protein 78 (GRP78) and glucose-regulated
protein 94 (GRP94). Furthermore, it decreased p-PERK, p-IRE1, and ATF6
Bisphenols are widely produced organic compounds worldwide [1]. They are applied in the production of plastic bottles, food packaging, inner coatings of beverage cans, thermal paper, and medical devices, among other products. These products can release monomers (such as bisphenol A (BPA)/S/C/F) under conditions such as heating, acidity, or alkalinity [1]. BPA monomers can enter the human body through the digestive system, vertical transmission between mother and fetus, respiratory system, and contact with the skin and eyes. BPA disrupts male reproductive function [2, 3]. BPA exposure can increase sperm DNA damage and decrease semen quality [4]. Additionally, BPA damages spermatogenesis and Sertoli cells in male rats [5, 6]. Consequently, the exposure to BPA has garnered considerable attention from both the public and the scientific community.
BPA can trigger apoptosis and endoplasmic reticulum (ER) stress of neuronal,
hepatic, and testicular cells [7, 8, 9]. BPA exerts a concentration-dependent effect
on the upregulation of ER stress pathway proteins in mouse testicular tissue.
Furthermore, BPA induces the upregulation of cleaved-caspase-3 in testicular
cells, leading to apoptosis [10]. Sulforaphane can target the ER stress signaling
pathway in liver cells, thus alleviating lipid droplet accumulation and key
enzyme levels responsible for fat synthesis induced by BPA [11]. Nano-selenium
has been found to alleviate BPA-induced intestinal damage by inhibiting the
expression of ER stress signaling proteins such as protein kinase R-like ER
kinase (PERK), inositol-requiring protein 1-
Melatonin [13] can regulate the function of the reproductive system. In the
female reproductive system, it can enhance the total antioxidant capacity of the
ovary and delay ovarian aging by increasing the superoxide dismutase level [14].
It enhances the developmental ability of embryos after cryopreservation and
vitrified oocytes by capturing free radicals and improving a series of events in
oxidative stress [15, 16]. Furthermore, it can effectively alleviate the
developmental damage to ovarian oocytes by some insecticides, such as acetamiprid
[17], rotenone [18], and imidacloprid [19]. Additionally, melatonin also
regulates the male reproductive system by enhancing testosterone synthesis and
secretion, promoting testicular growth and development, and improving semen
quality [20, 21, 22]. Melatonin exhibits potent antioxidant properties, which
alleviate damage to testicular tissue caused by anticancer drugs [23, 24],
radiation therapy [25, 26], bisphenols [27, 28, 29, 30, 31, 32], herbicides [33], and heavy metals
[34, 35]. Melatonin, when combined with bisphenol, enhances sperm viability,
density, and testosterone content [27, 29]. Both vitamin E and melatonin
effectively mitigate reproductive damage caused by bisphenols [27, 36]. Melatonin
is considered a more suitable agent for combating the toxicity of bisphenols [1].
Melatonin can alleviate ER stress in various tissues and cells [27, 28, 29]. It can
relieve ER stress and apoptosis in chondrocytes caused by osteoarthritis, by
inhibiting the IRE1-
Here, the impact of melatonin on testicular apoptosis and ER stress caused by BPA was investigated. Sexually mature male mice were simultaneously intervened with both BPA and melatonin. The testicular tissue development, the expressions of ER chaperone proteins and ER stress-related signaling pathway proteins, and testicular cell apoptosis were evaluated. Our findings may provide a novel therapeutic approach to alleviate ER stress in testicular cells and attenuate testicular injury.
Male sexually mature Kunming mice (weighing 35 g
The 20 mg/mL of BPA (239658, Sigma-Aldrich, St. Louis, MO, USA) was prepared by dissolving in olive oil. The 8 mg/mL of melatonin (M5250, Sigma-Aldrich) (0.24 g) was prepared by dissolving in 3 mL of anhydrous ethanol and subsequently 27 mL of normal saline to achieve a concentration of 8 mg/mL. The melatonin solution contained 10% ethanol, which has been recognized as non-toxic [37].
Thirty male mice were assigned randomly to the control, BPA, and BPA+melatonin groups. The BPA (50 mg/kg, a dose considered as the lowest-observed-adverse-effect level [38]), was administered to the BPA and BPA+melatonin groups daily by gavage. The control mice were administered an equivalent volume of olive oil daily. Meanwhile, the BPA+melatonin group was given 20 mg/kg melatonin (i.p.) every other day [39, 40]. The every-other-day administration approach can attenuate the stress damage induced by daily handling and injections in mice. Meanwhile, normal saline (equivalent volume) was given to the control and BPA groups. The mice in all three groups were treated with the respective interventions for 30 days, after which they were euthanized by cervical dislocation to obtain their testes. The right testis of each mouse was fixed, while the left testis was used for western blotting and enzyme-linked immunosorbent assay (ELISA).
Following 24 of fixation in Bouin’s fixative solution (BL-GO16, Nanjing Sumberger Biotechnology Co., Ltd., Nanjing, Jiangsu, China), the testis tissue was subjected to gradient ethanol dehydration, xylene treatment for 15 min, and paraffin embedment. Then, the tissue sections were subjected to treatment with xylene and graded ethanol. Followingly, staining with hematoxylin and eosin was performed. Finally, after dehydration and xylene treatment, the sections were examined using the Olympus BX53 microscope (Tokyo, Japan).
Mouse testicular tissues were incubated with RIPA lysis buffer and 1% PMSF
(P0013B and P1006, Beyotime, Beijing, China) to extract proteins. After protein
concentration determination, electrophoresis, and transfer, we conducted
incubation with primary antibodies against rabbit melatonin receptor 1A (MTNR1A)
(ab87639, Abcam, Cambridge, UK), melatonin Receptor 2 (MTNR2) (ab155678, Abcam),
GRP78 (ab21685, Abcam), GRP94 (ab238126, Abcam), p-PERK (3179,
CST, Boston, MA, USA), p-EIF2
Testicular cell apoptosis was evaluated with the Terminal Deoxynucleotidyl
Transferase dUTP Nick end Labeling (TUNEL) cell apoptosis detection kit (MK1024,
Wuhan Boster Biotechnology Co., Ltd., Wuhan, Hubei, China). The specific
procedure was as follows: the paraffin sections of testicular tissue were
de-parafinized, followed by gradient ethanol treatment. After proteinase
treatment for 10 min at 37 °C, the TUNEL reaction was performed through
treating the sections with terminal deoxynucleotidyl transferase and
digoxigenin-labeled dUTP for 2 h at 37 °C, which labeled the 3
After homogenizing the mouse testicular tissue and centrifugation, we quantified the testosterone in the supernatant with a testosterone detection kit (YJ001948, Shanghai Yuanju Biotechnology Center, Shanghai, China). The OD450 was determined utilizing a microplate reader. The testosterone content was quantified using the standard curve.
SPSS 26.0 (http://www.spss.com.hk/software/statistics)
evaluated the data, which are described by mean
The testes were fixed and stained with HE. In the BPA group, the epithelium of the seminiferous tubules in mouse testicular tissues became thinner. There was a reduction in germ cell number, disordered arrangement, and shedding of the germinal epithelium into the lumen (Fig. 1). In the control and BPA+melatonin groups, the layers of germinal epithelium were increased, with an orderly arrangement of germ cells, and no shedding of germinal epithelium was observed (Fig. 1). Therefore, melatonin mitigated the damage to mouse testicular tissues induced by BPA.
HE staining of mouse testicular tissue. HE staining results of the control, BPA, and BPA+melatonin groups are presented (Scale bar = 100 µm). The thickness of the spermatogenic epithelium is indicated by the double-headed arrow. The shedding of germinal epithelium is represented by the red arrow. The disordered arrangement of germ cells is indicated by the blue arrow. HE, Hematoxylin-Eosin; BPA, bisphenol A.
We assessed the expression of melatonin receptors MTNR1A and MTNR2 by using
western blotting (Fig. 2). Both MTNR1A and MTNR2 expressions in the BPA and
BPA+melatonin groups were significantly lower than the control (p
Expression of MTNR1A and MTNR2 in mouse testicular tissue.
MTNR1A and MTNR2 expression in mouse testicular tissue was assessed with western
blotting. The relative expression levels of MTNR1A and MTNR2 were compared after
grayscale analysis.
Compared to the control, the BPA and the BPA+melatonin groups exhibited
significantly elevated GRP78 and GRP94 in the testicular tissues, which are ER
molecular chaperones (Fig. 3) (p
Expression of molecular chaperones GRP78 and GRP94 in mouse
testicular tissue cells. GRP78 and GRP94 expressions in mouse testicular tissue
were measured with western blotting. Their relative expression levels were
compared after grayscale analysis (N = 3).
The induction of ER stress principally comprises IRE1, ATF6, and PERK signaling
pathways [41]. The protein expression levels of these pathways were assessed
through Western blot analysis (Fig. 4A,B). Both the BPA and BPA+melatonin groups
had significantly elevated levels of p-PERK, XBP-1, p-IRE1, p-EIF2
Expression of PERK, IRE1, and ATF6
Western blotting revealed that both the BPA and BPA+melatonin groups exhibited
significantly elevated expression of p-Caspase-3 and Caspase-12 than the control
(Fig. 5) (p
Expression of apoptotic proteins related to endoplasmic
reticulum stress in mouse testicular tissue. Western blotting detected cleaved
caspase-3 and caspase-12 in mouse testicular tissue. Their relative expression
levels were compared after grayscale analysis.
TUNEL assay was employed to detect apoptosis in mouse testicular cells. Cells
emitting green fluorescence indicate the presence of DNA fragmentation. Apoptosis
primarily occurred in spermatogonia and interstitial cells of mouse testicular
cells (Fig. 6A). The BPA and BPA+melatonin groups had significantly more
apoptotic testicular cells than the control (p
Detection of apoptosis in mouse testicular tissue. (A)
Apoptosis in mouse testicular tissue was detected using TUNEL. Green fluorescence
indicates apoptotic cells (Scale = 100 µm). (B) The apoptotic testicular
cell number was statistically analyzed.
Collectively, apoptosis in mouse testicular tissues after treatment with BPA is attenuated by melatonin.
The expression of the ER-related steroidogenic enzymes CYP17A1,
17
Expression of endoplasmic reticulum-associated steroidogenic
enzymes in mouse testicular tissue. The expressions of CYP17A1,
17
Testosterone content in mouse testicular tissue. The content of
testosterone in mouse testicular tissue was measured using enzyme-linked
immunosorbent assay (ELISA).
In recent decades, both humans and animals have been increasingly exposed to BPA, bisphenol S, bisphenol C, bisphenol F, bisphenol AF, and similar compounds [42, 43]. BPA and its analogs, due to their heat resistance, high fracture resistance, and electrical resistance, are utilized in the production of polycarbonate plastics and epoxy resins as cross-linking chemicals. Currently, extensive experimental, epidemiological, and clinical research is focusing on the safety of bisphenol compounds, especially BPA, which exhibits estrogen-like activity and serves as an endocrine disruptor [44, 45, 46]. People are gradually substituting BPA with analogs such as bisphenol AF in products [47]. BPA can accumulate in the bodies of males or male animals, leading to testicular tissue damage, affecting the development of seminiferous tubules, as well as the synthesis and secretion of testosterone [42, 48]. BPA can also cause ER stress in tissue cells, leading to cellular apoptosis, which is also an important pathway for BPA-induced testicular cell damage [49, 50]. Importantly, it has been found that antioxidants can reverse reproductive damage caused by environmental pollutants and protect the reproductive system [51]. Melatonin, with powerful antioxidant capabilities, can suppress the expression of ATF6, IRE1, and PERK proteins, which are related to the signal pathway of ER stress, thereby alleviating heat stress-induced cell apoptosis [23, 52]. Our study explored the function of melatonin against BPA-caused ER stress in mouse testicular cells. The results demonstrated suppressed the PERK, IRE1, and ATF6 signal pathways of ER stress, reduced testicular cell apoptosis, increased testosterone levels, and protected against the BPA-caused damages.
The unfolded protein response (UPR) is activated to rectify the issue of protein
folding defects. Despite this, its prolonged and excessive activation overwhelms
its protective effects, resulting in impaired ER function/homeostasis, cellular
damage, and apoptosis [41]. ER stress upregulates GRP78 and GRP94, which are
glycoproteins residing in the ER. Their activation regulates apoptosis triggered
by toxic injury [41]. Furthermore, ER stress triggers the activation of three
primary signaling pathways within the ER membrane: PERK, IRE1, and ATF6, which
then initiate downstream gene transcription to alleviate ER stress [41]. ER
stress can arise from exposure to various factors, including drugs [53],
environmental pollutants [54], and toxic substances [55]. BPA exposure can cause
ER damage in mouse oocytes, hepatocytes, and endometrial stromal cells, leading
to elevated levels of CHOP, p-EIF2
Melatonin, an important antioxidant, alleviates ER stress in spermatogonial stem
cells and testicular stromal cells by binding to the MTNR1A and MTNR2, thereby
down-regulating p-PERK, Xbp-1, p-IRE1, and GRP78 [58, 59]. Furthermore, melatonin
can reduce the expression of p-EIF2
Cell apoptosis acts as a protective mechanism to maintain organism homeostasis [62]. Caspase-12 regulates ER stress-induced cell apoptosis. Excessive ER stress can activate caspase-12, while ER stress-independent apoptosis does not involve this step [63]. The expression of caspase-12 can distinguish whether cell apoptosis is triggered by mitochondrial stress or ER stress. ER damage triggers the activation of procaspase-12, leading to its cleavage, conversion, and activation into caspase-12. Activated caspase-9 is subsequently cleaved into procaspase-3 and other effector caspases, ultimately leading to cell apoptosis [64]. Caspase-3 and caspase-12 are two critical proteins involved in ER stress-induced cell apoptosis. BPA upregulates caspase-3 and caspase-12 proteins, causing apoptosis in mouse brain cells and non-parenchymal liver cells [9, 65]. Melatonin suppresses cleaved caspase-3 in spermatogonial stem cells, thus preventing spermatogonial stem cell apoptosis [66]. Consistently, we demonstrated that melatonin suppressed BPA-induced cleaved caspase-3, caspase-12, and cell apoptosis, and protected testicular tissues.
Interstitial cell apoptosis is the predominant form of testicular cell apoptosis
induced by BPA [67]. Testicular interstitial cells are critical for testosterone
synthesis and release [68]. Prolonged exposure to BPA leads to reduced
testosterone synthesis and secretion [67]. The ER housing key enzymes like
CYP17A1, 3
As a potent antioxidant, melatonin enhances the expression of MTNR1A and MTNR2 on the membrane of testicular cells, inhibits ER stress and apoptosis caused by BPA, improves the expression of testosterone synthases on the ER, promotes testosterone synthesis and secretion, and protects the normal structure of testicular seminiferous tubules. Melatonin shows promise as a drug for preventing and treating oxidative stress-induced injury in testicular tissue in clinical practice. However, elucidating the mechanism by which melatonin inhibits ER stress in testicular cells is challenging and requires further investigation in subsequent cell experiments.
BPA, Bisphenol A; ER, endoplasmic reticulum; MTNR1A, melatonin receptor 1A;
MTNR2, melatonin Receptor 2; GRP78, glucose-regulated protein 78;
p-EIF2
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
GW and XP designed the study. QQ, LF, JL, and DX collected the data and performed the statistical analysis. QQ and XP analyzed and interpreted the data. QQ and LF collected the funds. QQ was a major contributor to manuscript writing. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.
The experimental procedures involving the mice were approved by the Ethics Committee of Jilin Medical University (No. 2023-LW013). The study is reported following ARRIVE guidelines.
Not applicable.
This research was funded by the Scientific and Technological Research Project of Jilin Province, grant number YDZJ202301ZYTS125, and the Graduate Innovation Plan Project of Beihua University, grant number 2022060.
The authors declare no conflict of interest.
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