Melatonin Relieves Busulfan-Induced Spermatogonial Stem Cell Apoptosis of Mouse Testis by Inhibiting Endoplasmic Reticulum Stress

Advances in chemotherapy for cancer treatment in young patients have resulted in increased survival and cure rates [1]. However, fertility is often impaired by alkylating agents, such as busulfan, which is a commonly used chemotherapeutic drug [2-4]. Therefore, the quality of life and the preservation of fertility have emerged as significant issues [5]. New methods should be developed to prevent testicular damage induced by alkylating agents [6, 7].

The endoplasmic reticulum (ER) is an intracellular organelle that controls protein folding, intracellular calcium homeostasis, and lipid synthesis [8]. Environmental stresses and increased protein synthesis often lead to the accumulation of unfolded proteins in the ER, and this phenomenon is called ER stress (ERS). The ERS pathway consists of three different branches: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase RNA-like ER kinase (PERK) [9].

ERS performs a dualistic function. It can restore ER homeostasis, but if ERS persists chronically at high levels, cellular functions can be negatively affected [10]. Chronic ERS is considered a key contributor to numerous human diseases, such as diabetes and neurodegeneration [11, 12]. Hence, targeting the components of ERS has been employed as a therapeutic strategy to control these ERS-associated pathologies [13, 14]. The IRE1 signalling pathway protects against ERS and controls the development and survival of immune cells [15]. ATF6 activates the expression of a repertoire of antioxidant genes and serves an important role as a link between the ERS and oxidative stress gene programmes [16]. Tuberous sclerosis complex-1 ablation induces prominent endoplasmic ERS responses by activating the PERK signalling pathways [17]. Moreover, ERS can also be induced by alkylating agents in renal epithelial cells [18].

Melatonin is secreted by the pineal gland, which is regulated by dark-light and seasonal cycles in mammals [19]. It acts as an antioxidant by reducing oxidative stress and increasing the activity of antioxidant enzymes in the body [20]. Melatonin protects melanocytes from DNA damage and oxidative stress through activation of NRF2 and its target enzymes [21]. It is well reported that melatonin can ameliorate ischaemic brain injury, preserve the functional integrity of the blood-brain barrier, and induce neurogenesis and cell proliferation through a receptor-dependent mechanism [22].

Melatonin is also involved in ER homeostasis [23]. It enhances the expression of the ERS proteins ATF6 and CHOP, improves survival rates, and successfully attenuates liver injury [24]. Furthermore, melatonin treatment alleviates ERS during bleomycin-induced pulmonary fibrosis [25]. Melatonin affects the regulation of testicular development and male reproduction by stimulating the secretion of gonadotropins and testosterone [26]. It also prevents cisplatin-induced primordial follicle loss [27]. In addition, the research recently published by our group has reported that melatonin has beneficial effects on oxidative apoptosis of mouse SSCs induced by busulfan [28]. Therefore, we aim to assess whether ERS is involved in busulfan-induced SSC apoptosis and testicular damage. The present study will provide a theoretical basis for alleviating the side effects caused by busulfan in clinical cancer treatment.

In this study, the beneficial effects of melatonin on busulfan-induced testicular damage and mouse spermatogonial stem cell (SSC) apoptosis were demonstrated. Our results revealed that the presence of melatonin directly provided resistance to busulfan-induced apoptosis and correlated with ERS. In vitro and in vivo evidence demonstrated that the administration of melatonin could ameliorate testicular damage caused by busulfan. Therefore, melatonin might be useful as a potential drug to counteract the side effects of busulfan because of its advantages, including high efficiency, low toxicity and endogenous effects.

Experiments were performed unhealthy eight-week-old ICRmale mice weighing between 2 5 and 30 g. The mice were obtained from the animal centre of the Fourth Military Medical University and were housed in wire cages at 25°C in a 12 h/12 h light/dark cycle at 70% humidity and fed ad libitum. The mice were maintained and the experiments were completed in accordance with the Guide for the Care and Use of Laboratory Animals at Northwest A&F University.

In this experiment, busulfan and 4PBA were diluted in DMSO, and melatonin was dissolved in 1% ethanol (in normal saline). Eighty age-matched mice were randomly separated into four groups: the CON group received vehicle injection (DMSO), the BU group received a single busulfan treatment (30 mg/kg), the BU+MEL group received melatonin (10 mg/kg/day) after busulfan treatment for 4 weeks, and the BU+4PBA group received 4PBA (100 mg/kg/day) after busulfan treatment for 4 weeks.

C18-4 cells, which are type A spermatogonial stem cells, were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12, Gibco, Grand Island, NY) supplemented with 10% foetal bovine serum (FBS, Gibco), 2 mM of L-glutamine (Invitrogen, Carlsbad, CA), and 100 unit/ml of penicillin and streptomycin (Invitrogen). The cells were subcultured every 3 days and maintained at 37°C in an incubator humidified with 5% CO₂. The cells were dissociated by treating with 0.25% trypsin-EDTA (Invitrogen) and reseeded into multi-well plates for subsequent analysis. The medium was changed every 2 d. In this procedure, busulfan was dissolved in DMSO to 10^-4 µM. Melatonin was dissolved in 1% ethanol in physiological saline to 10^-7M. 4PBA was dissolved in DMSO to 1 mM. Cells were cultivated with busulfan (BU), busulfan and melatonin (BU+MEL), or busulfan and 4PBA (BU+4PBA) for 24 h prior to the experiments.

For histological analyses, testes were dissected from CON, BU, BU+MEL and BU+4PBA mice. After fixation in 4% PFA solution, the samples were embedded in paraffin blocks. Sections were cut and then stained with haematoxylin and eosin (HE).

For sperm morphological analyses, cauda epididymal sperm from CON, BU, BU+MEL or BU+4PBA mice were spread onto slides with PBS and air-dried. Sperm smears were stained with Giemsa.

Immunofluorescence staining was performed as described in our previous studies [29]. The details of antibodies used are listed in Table 1.

The testes were collected, and protein extracts were obtained after surgery. The proteins were quantified using a BCA protein assay kit (Beyotime, China). The proteins were separated through SDS–PAGE and subsequently transferred to Immobilon NC membranes (Millipore, Boston, MA, USA). The membranes were blocked with 6% non-fat milk in TBST at room temperature, incubated with primary antibodies (the details are listed in Table 1) overnight at 4°C and washed thrice with TBST for 15 min each. The membranes were incubated at 37°C for 1 h with appropriate secondary antibodies, including goat anti-rabbit IgG-HRP (1: 3000, Boster) and rabbit anti-mouse IgG-HRP (1: 2000, Boster) and then washed with TBST thrice for 15 min each time. Protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified using Quantity One (West Berkeley, CA, USA).

Total RNA was extracted from testes using the Trizol reagent (Qiagen, Beijing, China) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a Reverse Transcriptase Reagent kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. qRT-PCR analysis was conducted in triplicate onaCFX96 Real-time PCR system (Bio-Rad Ltd, Berkeley, CA, USA) according to the directions of the BioEasy SYBRGreen 1 RT-qPCR kit (Bioer Co. Ltd., Hangzhou, China). The qRT-PCR was performed as described previously [28]. Gapdh expression was used as the house-keeping control. The comparative CT method was used to measure the relative gene expression. The primers are listed in Table 2.

Cell cycle distribution was assessed by propidium iodide (PI) staining. Adherent cells were dissociated with 0.2 5% trypsinization. The samples were washed with PBS, stained with 50 µg/ml of PI and 50 µg/ml of RNase A at 37°C for 20 min and analysed through flow cytometry (Altra; Beckman Co.).

Cell apoptosis was evaluated through annexin V-FITC and PI staining. The cells for the apoptosis test were resuspended gently in 500 µl of PBS. Then, 5 µl of annexin V-FITC and 5 µl of PI were added to the medium and mixed gently. The cells were incubated at room temperature for 10 min in the dark and subjected to FACS analysis.

The intracellular Ca²⁺ level was measured by using the Fluo-3 AM (Beyotime, China). Cells (5 × 10⁵) were pretreated with BU, BU and MEL or 4PBA for 24 h, loaded with Fluo-3 AM for 1 h and then subjected to flow cytometric analysis.

Statistical analyses were performed using one-way ANOVA with the Newman–Keuls multiple ran test. Student’s t test was performed when only two pairs of data were compared. Data were represented the mean ± SD, and statistical significance was expressed as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. All data are representative of at least three different experiments and were analysed with GraphPad Prism (La Jolla, CA, USA).

To examine whether melatonin treatment influences there generation of spermatogenesis in the male mouse genital tract impaired by busulfan, we histopathologically analysed the testes and epididymis of different treatment groups. The results showed that the testicular mass of the busulfan-treated mice was reduced compared to control testes (48 ± 13% versus control group, P < 0.05, Fig. 1A). 4PBA works as a chemical chaperone by inhibiting ERS [30]. To determine whether the protective mechanism of melatonin is through suppression of ERS, we introduced 4PBA and found that the testìcular weight was significantly increased in the melatonin and 4PBA treatment groups compared to busulfan-treated testes (62 ± 15% and 68 ± 22% versus the busulfan-treated group, respectively, P < 0.05, Fig. 1B). Approximately 50% of the mice died roughly 30 days after they were treated with busulfan, but the death rate was only 17% and 22% in the melatonin- and 4PBA-treated mice, respectively (Fig. 1C).

No histopathological abnormalities were observed in the control testes, and almost all seminiferous tubules were undergoing complete spermatogenesis. The busulfan-treated testes consisted of thin seminiferous epithelia, large lumens, and testicular vein congestion. Most seminiferous tubules were devoid of germ cells, and numerous degenerating germ cells could be observed in the seminiferous tubules of the busulfan-treated testes. Melatonin significantly enhanced the spermatogenic recovery after busulfan treatment was administered (Fig. 1D). The proportion of tubules with spermatogenesis was declined in the busulfan treatment group compared to control testes (P < 0.05). However, the percentage of seminiferous tubule with spermatogenesis in testes of the melatonin- and 4PBA-treated group were increased than that of the busulfan-treated group (48 ± 28% and 57 ± 7% versus busulfan-treated group, respectively, P < 0.05, Fig. 1E). The quantitative data indicated that a significant number of germ cells lost in busulfan treatment group. Moreover, the total number of spermatogonial cells was drastically reduced in the busulfan treatment group compared to control group (74 ± 21%, P < 0.05, Fig. 1F). Exposure of busulfan decreased the number of normal sperm per epididymis compared to control mice (73 ± 13%, P < 0.05). The count of normal sperm was increased 2.2 and 2.4-fold in the melatonin- and 4PBA-rescued groups relative to the busulfan-treated group (P < 0.05, Figs. 1G-H). These results suggest that melatonin significantly prevented busulfan-induced testicular damage and spermatogonium loss.

To analyse the protective mechanism of melatonin for the testes against toxicity caused by busulfan, we used 4PBA and investigated the protein expression of the ERS indicators ATF6 and PERK in the testes. Consistent with histopathological findings, our results demonstrated that the intensity of the immunofluorescent signal for ATF6 and pIREl was markedly increased in the majority of busulfan-treated mice compared with the control mice (P < 0.05). The administration of melatonin or 4PBA significantly decreased the expression levels of ATF6 and pIREl (P < 0.05, Fig. 2A–D).

We also assessed the expression levels of ERS-related genes in the testes through qRT-PCR. Compared with that in the control group, the mRNA expression of GRP78, Atf6, pIREl, and Xbpl in the busulfan-treated group was significantly increased (0.14-, 2.26-, 2.99-, and 3.79-fold increases compared with the control group, respectively, P < 0.05). However, this increasing trend was significantly reversed when the treatment was combined with melatonin or 4PBA (P < 0.05, Fig. 2E).

Western blotting was applied to examine the effect of ERS activation. In the control group, a low and basal activation level of the ERS-related proteins was detected. After busulfan was added, the expression of GRP78, pIREl, XBP1, and ATF6 was clearly stimulated. In the presence of melatonin or 4PBA, the activation effect was reversed (P < 0.05, Fig. 2F–G). Therefore, melatonin protected the testes from ERS damage.

To evaluate the protective mechanism of melatonin‚ we investigated the protein expression of the ERS-related apoptotic genes CHOP and PUMA through an immunofluorescence assay. CHOP had a high level of expression after busulfan treatment but low expression in the melatonin- and 4PBA-treated groups (P < 0.05, Fig. 3A–B). Compared to control mice, intense fluorescent signals of PUMA were found in the busulfan-treated group. Conversely, weak signals were observed in the melatonin- and 4PBA-treated groups (P < 0.05, Fig. 3C-D). The mRNA expression levels of CHOP, PUMA, caspase-3 and caspase-12 were examined through qRT-PCR analysis, and similar to the immunofluorescence analysis, the mRNA expression level of ERS-related apoptotic genes was increased relative to the control group after busulfan was administered, whereas the increase was significantly compromised by melatonin or 4PBA (P < 0.05, Fig. 3E).

Western blotting results further revealed that co-administration of busulfan with melatonin or 4PBA significantly downregulated the expression of the ERS-mediated apoptosis protein CHOP, cleaved caspase-12, PUMA, and cleaved caspase-3, all of which were upregulated by busulfan (P < 0.05, Fig. 3F–G). Therefore, ERS might be activated by busulfan and act to further induce testicular cell apoptosis and spermatogonium loss. The combined treatment with melatonin abolished the adverse effects of busulfan on the testes.

To evaluate the importance of melatonin receptors (MT) in facilitating the therapeutic functions of melatonin, we performed an immunofluorescence analysis of melatonin receptors in the testes and mouse SSCs. The untreated cells showed normal signals of MT1 and MT2. However, only weak signals were found in busulfan-treated cells. In contrast, the fluorescent signals of MT1 and MT2 were highly enriched in melatonin- and 4PBA-treated cells (Fig. 4A–C). The quantitative results of the immunofluorescence assay showed that the positive signals were weaker in the busulfan-treated group than in the control cells. The fluorescent signals of MT1 and MT2 were enhanced when melatonin was administered compared with the busulfan-treated group. Moreover, 4PBA treatment increased the signals of MT1 and MT2 (P < 0.05, Fig. 4B–D). Similar to cell immunofluorescence analysis, testicular fluorescence analysis revealed that intense MT1 and MT2 fluorescent signals were observed in melatonin-or 4PBA-treated testes; however, weak signals were found in the seminiferous tubules of the testes treated with busulfan (Fig. 4E). The quantitation results confirmed that the MT1 and MT2 expression levels were increased in the melatonin-treated group compared with the busulfan-treated group. 4PBA administration increased MT1 and MT2 expression compared with that of the busulfan group (P < 0.05, Fig. 4F). Western blotting further confirmed that administration of melatonin significantly upregulated the expression of MT1 and MT2 (P < 0.05, Fig. 4G–H). These data suggested that the protective role of melatonin against mouse testicular damage was mediated by MT1 and MT2.

The mouse C18-4 cell line, which is a type A SSC, was examined to verify the direct effects of melatonin and busulfan on spermatogonial cells. To confirm the involvement of ERS, we co-treated the cells with 4PBA. Co-treatment with melatonin was also administered to assess the role of melatonin in busulfan-induced apoptosis. After treatment with busulfan, the morphological characteristics of the C18-4 cells were shrunken, and their surface was rough in comparison to the control cells, as revealed by light microscopy. Conversely the co-administration of melatonin or 4PBA significantly reversed these morphological changes (Fig. 5A). We counted the number of annexin V FITC-positive cells through flow cytometry analysis. Compared to the control group, busulfan treatment increased the percentage of apoptotic cells; however, the administration of melatonin or 4PBA decreased the percentage of apoptotic cells compared with the busulfan-treated cells (P < 0.05, Fig. 5B–C). To confirm these results, we analysed the apoptotic proteins P53 and cleaved caspase-3 through immunofluorescence assay. Intense fluorescent signals indicating the presence of apoptosis were observed in the busulfan-treated cells. Conversely, only slight fluorescence was detected in the melatonin- and 4PBA-treated cells (Fig. 5D-E). These experiments suggested that melatonin treatment could sufficiently enhance the resistance of mouse SSCs to busulfan-induced apoptosis.

Cell cycle distribution was measured to explore the effects of melatonin and busulfan on the proliferation of mouse SSCs. Our results indicated that the percentage of busulfan-treated cells in the S phase was reduced compared with the control group. In contrast, the percentage of S-phase cells increased in the melatonin- and 4PBA-treated cells (P < 0.05, Fig. 6A–B). Cell proliferation was observed by using the EdU kit. The cell proliferation rate was reduced in the busulfan treatment group compared with that of the control group (18.5 ± 9%, P < 0.05). Conversely, melatonin promoted the proliferation activity of mouse SSCs. Compared with the busulfan-treated group, the proliferation of the SSCs was increased in the 4PBA-treated group (P < 0.05, Fig. 6C–D). To confirm these results, we analysed the proliferative protein PCNA through immunofluorescence assay and found that the number of positive cells was reduced in the busulfan-treated group relative to the control cells (P < 0.05). In contrast, the percentage of PCNA-positive cells in the melatonin- and 4PBA-treated group increased (P < 0.05, Fig. 6E–F). These data suggest that melatonin may promote SSC survival and drive their proliferation to promote regeneration after busulfan treatment.

C18-4 cells were examined through immunofluorescence assays to clarify whether spermatogenic cells treated with busulfan in vitro induced ERS and to determine the effect of melatonin on the expression of ERS-related molecules. The ERS indicator ATF6 had a high level of expression in the busulfan group but low expression in melatonin- and 4PBA-treated cells (P < 0.05, Fig. 7A, D). Cell imaging revealed that the pIREl signals were enhanced in the busulfan group relative to the control group. In comparison, this strong signal was suppressed by either melatonin or 4PBA (P < 0.05, Fig. 7B, E). Intense fluorescent signals of pPERK indicated that higher ERS levels occurred in the busulfan-treated cells than in the control group. Conversely, low levels of fluorescence showed that the ERS level was reduced in melatonin- and 4PBA-treated cells compared with the busulfan treatment group (P < 0.05, Fig. 7C, F). We also measured intracellular Ca²⁺ levels through Fluo-3AM staining because ER is a major calcium-storage organelle, and its calcium content is essential for proper protein folding and transport. The disruption of calcium homeostasis can disturb ER functions and induce ERS [31]. Our results indicated that the intracellular Ca²⁺ concentration increased in C18-4 cells treated with busulfan. Conversely, the co-treatment of busulfan with melatonin or 4PBA failed to increase intracellular Ca²⁺ levels (Fig. 7G). Therefore, busulfan enhanced the activation of ERS in C18-4 cells, but co-treatment with melatonin resulted in improved ER homeostasis.

To obtain insights into the mechanism by which busulfan induces apoptosis and to clarify whether melatonin prevents apoptosis caused by ERS in mouse SSCs, we performed immunofluorescence analysis. The results demonstrated that the signals from the ERS-related apoptotic marker cleaved caspase-12 were enhanced in the busulfan-treated cells compared with the control cells. In contrast, a relatively weak fluorescence signal was detected in the cells treated with busulfan combined with melatonin or 4PBA (P < 0.05, Fig. 8A–B). The quantitative results of the immunofluorescence assay of whole cells revealed that the CHOP expression level was increased in the busulfan-treated cells relative to the control cells. Conversely, the signal was suppressed when the cells were co-treated with melatonin or 4PBA (P < 0.05, Fig. 8C–D).

We also investigated PUMA expression, which is reported to be a proapoptotic gene induced by ERS [32]. Our results showed that melatonin suppressed PUMA expression stimulated by busulfan (P < 0.05, Fig. 8E–F). From these results, we conclude that mouse SSCs exposed to busulfan undergo ERS-related apoptosis, but in contrast, cells co-administered melatonin or 4PBA conferred higher resistance than busulfan-treated cells.

Chemotherapeutic drugs, such as busulfan, are often used to treat cancer and increase the survival rates of patients. However, these chemotherapeutics may induce temporary or permanent reproductive toxicity in male patients. Children and adolescents with testicular cancer or leukaemia are at high risk of developing permanent sterility because of chemotherapeutic application. Therefore, strategies that effectively preserve fertility during the course of cancer treatment should be developed [33, 34]. In this study, we provide evidence that melatonin is sufficient to counteract busulfan-induced testicular damage and SSC apoptosis. Moreover, we found that the protective effect of melatonin is dependent on ERS alleviation.

The ER is an important organelle involved in protein folding calcium homeostasis maintenance, and lipid biosynthesis [35]. ERS occurs when unfolded proteins accumulate and the folding capacity of ER chaperones exceeds the capacity of the ER lumen to facilitate their disposal. It is a major contributor to various diseases, such as cancer, diabetes and Alzheimer’s disease [36, 37]. As such, targeting the components of the ERS signal pathway has been widely explored as a therapeutic strategy [38].

Despite a large number of studies linking ERS to toxicants, currently, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage and apoptosis [39, 40]. In this study, we demonstrated the relationship between testicular damage and ERS pathways in response to busulfan. We evaluated ERS-related genes and found an increased expression of GRP78, ATF6, pIREl, and XBP1 in busulfan-treated mouse testes. This finding suggested that ERS could be responsible for busulfan-induced testicular damage. A recent report showed that activation of ATF4 and CHOP could enhance protein synthesis and subsequently induce ERS and apoptosis [41]. Our results further demonstrated that busulfan treatment increased the expression of ERS-related apoptotic proteins, such as CHOP and cleaved caspase-12. We also found an increase in P53 and PUMA protein levels in response to busulfan treatment, which could be further confirmed by evaluating levels of apoptosis because P53 acts as a transcription factor that regulates the expression of apoptosis-related genes [42], and PUMA is a potent pro-apoptotic protein because of its ability to bind and neutralize all pro-survival BCL2 members [43]. Therefore, ERS regulation could involve apoptotic actions in testicular damage. Our experiments on mouse SSCs were consistent with that on the mouse testes. These findings (both in vitro and in vivo) suggest that inhibition of ERS might be a target of novel therapeutics for male infertility.

Melatonin and its metabolites are well-known broad-spectrum antioxidants and free radical scavengers that regulate various molecular pathways, such as inflammation, proliferation, apoptosis, and metastasis, during different pathological conditions [44-46]. It has been documented that melatonin modulates the signalling of neuronal cell death [47] and bladder cancer cells induced by ERS [48]. Melatonin significantly reverses tunicamycin-induced ERS in human hepatocellular carcinoma cells and improves cytotoxic responses to doxorubicin by increasing the expression of CHOP [49]. A couple of studies revealed that melatonin alleviates Cd-induced cellular stress and germ cell apoptosis in testes [50] and prevents bisphenol A-induced apoptosis by controlling BCL2 expression and ameliorating oxidative stress in the testes and sperm [51]. Furthermore, melatonin had a positive influence on the efficiency of male germ cell differentiation [52], and it can promote goat SSC proliferation by stimulating GDNF production in Sertoli cells [53].

Similarly, our data showed that melatonin contributed to the proliferation of mouse SSCs, but the proliferation was suppressed when they were exposed to busulfan. Indeed, our recent study has shown that melatonin markedly reduces the extent of testicular injury by inhibiting oxidative stress [28]. However, whether melatonin could relieve busulfan-induced ERS in testes and SSCs was previously unclear. Here, we showed that melatonin significantly alleviated busulfan-induced testicular ERS by suppressing the expression levels of the key ERS genes pIREl and ATF6 and inhibiting the ERS-related apoptotic genes CHOP and caspase-12. Moreover, the integrity of the testes was significantly preserved by melatonin treatment. Previous studies reported that the effects of melatonin were mediated by the specific receptors MT1 and MT2 [54]. Melatonin pretreatment inhibited the activating role of tunicamycin on ERS through melatonin receptor-mediated IRE-1 signalling in skeletal muscle cells [55]. Similarly, our study suggests that the expression of MT1 and MT2 was increased in exogenous melatonin-treated mouse testes and SSCs, and these findings imply that the protective role of melatonin against mouse testicular damage could be exerted through MT1 and MT2.

4PBA is known to be a major repressor of ERS by improving the folding capacity of misfolded proteins [30]. It is capable of counteracting the toxic effect of proteinuria through suppressing ATF4 and attenuating ERS in chronic kidney disease [56]. To elucidate the mechanism behind the inhibition of ERS by melatonin, 4PBA was utilized. We found that the expression of ERS-related genes and apoptosis genes decreased to normal levels when 4PBA was co-administered with busulfan. Similar to 4BPA, melatonin is able to protect against cerebral IR injury through inhibiting ERS via PERK and IRE1 signals [57]. Downregulation of ATF-6 by melatonin contributes to ERS-induced apoptosis of human hepatoma cells [58]. In the current study, we found that melatonin could abrogate busulfan-induced ERS in mouse SSCs by inhibiting the ERS-related genes IRE1 and PERK, which further depressed pro-apoptotic transcript CHOP activation, then inhibited P53 expression and resulted in the reduction of PUMA, and consequently decreased the accumulation of cellular caspase, thereby preventing cell apoptosis (Fig. 9). Thus, we conclude that melatonin treatment significantly protected mouse SSCs from busulfan-induced apoptosis and protected testes from damage in busulfan-treated mice. This study suggests that melatonin could provide potential therapeutic advantages by protecting the testes from busulfan-induced injury.

Taken together, our study demonstrated that the activation of ERS signalling contributed to busulfan-induced SSC apoptosis and that melatonin treatment significantly protected mouse SSCs from busulfan-induced apoptosis. This study provides basic information that helps us to understand the recovery of spermatogenesis in men after cancer treatment and provides guidance about the possible effects that new agents might have on the testes of male patients with cancer.

This work was supported by grants from the National Key Research and Development Program of China (2016YFA0100203 and 2016YFD0500306), the General Programs of Natural Science Foundation of China (31572399, 31272518), and the Key Project of Shaanxi Province of Science and Technology (2015NY157).

The authors declare no conflicts of interest.