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. 2026 Aug 28;40(5):2844–2854. doi: 10.21873/invivo.14434

Ramelteon Mitigates Cisplatin-induced Testicular Dysfunction in Rats

SHUN KAWAMURA 1, KOJI CHIBA 1, KAZUKI TSUBOTANI 1, CHANHYON KIN 1, YOSUKE YAMASHITA 1, KATSUYA SATO 1, KEISUKE OKADA 1, HIDEAKI MIYAKE 1
PMCID: PMC13531113  PMID: 42665423

Abstract

Background/Aim

Cisplatin is a widely used anti-cancer agent, but it can cause testicular dysfunction, leading to infertility and hypogonadism. Currently, pharmacological agents for preventing testicular dysfunction during chemotherapy are limited. Ramelteon, an insomnia medication that selectively targets melatonin receptor type 1 and type 2, exerts cytoprotective effects across organs via anti-inflammatory and anti-apoptotic mechanisms. Ramelteon has an established safety profile in humans; however, its efficacy against chemotherapy-induced testicular toxicity remains unclear. We therefore examined the protective efficacy of ramelteon against cisplatin-induced testicular dysfunction in a rat model.

Materials and Methods

Male 8-week-old Jcl:Wistar rats were randomized to control (saline+vehicle), ramelteon (10 mg/kg/day orally on days 1-7), cisplatin (7 mg/kg intraperitoneally once on day 1), or cisplatin+ramelteon (n=8 per group). On day 8, blood, testes, and epididymides were collected. Endpoints included sperm parameters, testicular histopathology, Ki-67 immunostaining, serum testosterone, and testicular quantitative polymerase chain reaction (qPCR) for steroid synthesis-related (Star, Cyp11a1, Cyp17a1, Hsd17b3) and apoptosis- and inflammation-related (Casp3, Il6) transcript levels.

Results

Cisplatin administration induced histological damage to the seminiferous tubules, reduced sperm counts and motility, decreased testosterone levels, increased testicular interstitial Ki-67 positivity, suppressed steroid synthesis-related gene expression, and increased apoptosis-related and inflammation-related gene expression. Co-administration of ramelteon significantly attenuated these changes, resulting in reduced pathological tissue damage in the testes, attenuation of deterioration in sperm parameters and testosterone levels, and improvement in steroid synthesis-related and inflammation-related gene expression.

Conclusion

Ramelteon mitigated acute cisplatin-induced testicular damage in rats by attenuating deterioration in sperm parameters and steroidogenesis-related changes. These findings suggest that ramelteon may be a candidate for further investigation as a strategy to mitigate acute chemotherapy-induced testicular injury.

Keywords: Cisplatin, ramelteon, testicular toxicity, spermatogenesis, steroidogenesis

Introduction

The platinum agent cisplatin (CDDP) is commonly utilized to manage several solid malignancies, notably testicular, lung, and ovarian cancers (1). However, testicular toxicity is one of the principal adverse effects of CDDP, posing serious long-term risks, such as infertility and hypogonadism, especially in young male patients (2). In fact, in studies of male patients who received CDDP-based chemotherapy for testicular cancer, only 41%-57% had normal semen parameters more than 1-2 years after treatment, with 23%-24% exhibiting oligospermia and 20%-35% experiencing azoospermia (3-5). Although spermatogenesis can recover during long-term follow-up, the recovery is often incomplete. The pregnancy success rate among men within 12 years after four courses of bleomycin-etoposide-CDDP therapy was only 76%, and the median time to first childbirth was 6.4 years, suggesting that significant time is required for spermatogenic function to improve following CDDP administration (6). Additionally, many male patients who underwent CDDP-based chemotherapy exhibited Leydig cell dysfunction, suggesting mild gonadal dysfunction (7). Animal studies have also demonstrated that CDDP impairs testicular structure, suppresses steroid production, and reduces sperm quality and testosterone levels (8-11). In addition, recent preclinical evidence has suggested that contemporary cancer therapies, including immune checkpoint inhibitors, may also affect reproductive function, underscoring the broader importance of evaluating cancer therapy-related gonadal toxicity (12). Beyond gonadal toxicity, CDDP-related adverse effects remain clinically important because they can affect treatment completion and dose intensity, as shown in recent clinical studies focusing on renal dysfunction during CDDP-based chemotherapy (13, 14).

CDDP-induced testicular dysfunction arises from multiple pathophysiological processes, including oxidative stress driven by reactive oxygen species overproduction, reduced testosterone secretion associated with Leydig cell dysfunction, germ cell and Leydig cell apoptosis, mitochondrial dysfunction, and activation of inflammatory responses attributable to increased expression of tumor necrosis factor-α and inducible nitric oxide synthase (8-11). Therefore, drugs with antioxidant or anti-apoptotic effects have been considered candidates for preventing CDDP-induced testicular toxicity. Melatonin is one such candidate, with animal studies suggesting its ability to protect against CDDP-induced testicular damage and improve spermatogenesis and testosterone secretion (15-17). These effects are attributable to both direct free-radical scavenging and receptor-mediated signaling pathways, with diverse actions including antioxidant, anti-apoptotic, and anti-inflammatory effects (18). However, despite the evaluation of multiple agents, including melatonin, as potential protective agents for testicular function in animal experiments, no drugs have been used clinically in humans to date.

Ramelteon, a melatonin agonist that selectively acts on melatonin receptor type 1 and type 2 (MT1/MT2), is approved for use in humans as a treatment for insomnia (19). Unlike melatonin, it does not possess direct antioxidant activity (20), but it has high receptor affinity and a long half-life (21, 22). In recent years, the cell-protective effects of ramelteon have garnered attention. In basic research, ramelteon protected against hepatic ischemia-reperfusion injury, myocardial infarction, and methotrexate-induced pulmonary toxicity through anti-apoptotic and anti-inflammatory actions (20, 23, 24). However, its efficacy against chemotherapy-induced gonadal dysfunction has not been sufficiently investigated to date. Whether existing approved drugs such as ramelteon can mitigate CDDP-induced gonadal toxicity remains an important research question. Thus, the present study assessed the capacity of ramelteon to ameliorate CDDP-induced testicular dysfunction in rats.

Materials and Methods

Drugs. CDDP and ramelteon were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). CDDP was dissolved in 0.9% saline and delivered intraperitoneally at 7 mg/kg. Ramelteon was dissolved in dimethyl sulfoxide, suspended in corn oil, and administered orally at 10 mg/kg. The dosage and administration schedule of ramelteon were selected based on previous reports (19, 24, 25).

Animals and experimental protocol. The experimental protocol was approved by the Animal Care and Use Committee of Kobe University (approval number: P231006) and the study followed institutional guidelines for the care and use of laboratory animals. Male Jcl:Wistar rats (8 weeks old, 220-280 g; CLEA Japan Inc., Tokyo, Japan) were housed under standard conditions with free access to water and food on a 12 h light/dark cycle, with lights on at 6:00 a.m., and acclimated for 1 week prior to the experiment. After the acclimatization period, rats showing no apparent abnormalities in appearance or behavior were included in the study. The sample size was set based on previous animal studies using cisplatin-induced testicular dysfunction models, while minimizing animal use and allowing statistical comparisons among groups (8, 11, 26). A total of 32 eligible rats were allocated to the following four groups by block randomization using a computer-generated randomization sequence (n=8 per group): (i) control group (Ctrl; saline+vehicle); (ii) ramelteon group (RMT; saline+ramelteon 10 mg/kg/day); (iii) CDDP group (CDDP 7 mg/kg+vehicle); (iv) CDDP+ramelteon combination group (C+R; CDDP 7 mg/kg+ramelteon 10 mg/kg/day). On day 1, rats in groups 3 and 4 received a single intraperitoneal injection of CDDP, whereas the other rats received physiological saline. On days 1-7, ramelteon or vehicle was administered by oral gavage once daily immediately before the onset of the dark phase, considering the circadian rhythm of endogenous melatonin secretion (27, 28). On day 8, all rats were euthanized between noon and 1:00 p.m. with an overdose of isoflurane followed by thoracotomy, and blood, testes, and epididymides were collected for analysis.

Evaluation of sperm parameters. After measurement of the whole left epididymal weight, the left cauda epididymis was used for sperm analysis. The cauda epididymis was incised twice in 1 ml of 0.9% saline to prepare a suspension, which was incubated for 10 min at 37°C (26). A 10-µl aliquot was applied to a Makler sperm counting chamber, and the sperm count (1 × 106/ml) and motility rate (%) were measured. Motile and immotile spermatozoa were counted in randomly selected fields under light microscopy, and sperm motility rate was expressed as the percentage of motile spermatozoa among the total number of spermatozoa counted.

Histopathological analysis. The left testes of rats were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned into 4-µm-thick slices. The specimens were stained with hematoxylin and eosin (H&E) and observed at ×400 magnification using a BZ-X710 optical microscope (Keyence Co., Osaka, Japan). For quantitative assessment of seminiferous tubule diameter, 20 randomly selected round or nearly round seminiferous tubules per rat were measured. To minimize the influence of oblique sectioning, the shortest diameter passing through the center of each tubule was measured using ImageJ Fiji software (US National Institutes of Health, Bethesda, MD, USA) (29). The average value for each animal was used for statistical analysis.

Immunohistochemistry. Immunohistochemistry was performed using an automated tissue processing system (Bond-Max, Leica Microsystems, Wetzlar, Germany) according to standard protocols. Ki-67 expression (rabbit polyclonal antibody, dilution 1:200; Abcam, Cambridge, UK; #ab15580) was evaluated as a cell proliferation marker. Twenty random fields were photographed per sample at ×200 magnification, and the percentage of Ki-67-positive cells in the testicular interstitium was measured using ImageJ Fiji software.

Serum testosterone. Blood was obtained by cardiac puncture immediately after anesthesia induction. Blood testosterone levels were quantitatively measured using an electrochemiluminescence immunoassay performed by SRL (Tokyo, Japan).

Quantitative real-time PCR (RT-qPCR). Messenger RNA was extracted from testicular tissue using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The concentration and purity of RNA were determined using a NanoDrop One (Thermo Fisher Scientific). Complementary DNA was synthesized using 1 µg of total RNA and SuperScript IV VILO Master Mix (Thermo Fisher Scientific). RT-qPCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan Gene Expression Assays (Thermo Fisher Scientific) on an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific). The primers and probes used for RT-qPCR were as follows: Hsd17b3 (Rn00588942_m1), Cyp17a1 (Rn00562601_m1), Cyp11a1 (Rn00568733_m1), and Star (Rn00580695_m1) as steroidogenesis-related markers; Casp3 (Rn00563902_m1) as an apoptosis-related marker; Il6 (Rn01410330_m1) as an inflammation-related marker; and Actb (Rn00667869_m1) as the endogenous control. The expression of each gene was quantified relative to that in the control group using the ∆∆Ct method.

Statistical analysis. All values are reported as the mean ± standard error (SE). Statistical analyses were performed using EZR, a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (30). Normality was checked using the Shapiro-Wilk test for each endpoint. For parametric data, one-way analysis of variance and Tukey’s post hoc test were used. For non-parametric data, the Kruskal-Wallis and Steel-Dwass tests were used. A p-value <0.05 was considered statistically significant.

Results

Testicular and epididymal weights. Figures 1A and 1B present the weights of the excised testes and epididymides. There were no statistically significant differences in testicular and epididymal weights among the groups.

Figure 1.

Figure 1

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Reproductive organs and sperm parameters in each group. (A) Testis weight. (B) Epididymis weight. (C) Sperm count. (D) Sperm motility. Data are presented as the mean±standard error (SE); n=8 per group. Statistical analysis was performed using the Kruskal-Wallis test followed by Steel-Dwass test. *p<0.05, **p<0.01. Ctrl, Control group; CDDP, cisplatin group; RMT, ramelteon group; C+R, cisplatin plus ramelteon group.

Sperm parameters. Sperm parameters are presented in Figures 1C and 1D. The sperm count and motility were significantly lower in the CDDP group than in the Ctrl and RMT groups (sperm count: p<0.01; motility: p<0.05). The sperm count was significantly higher in the C+R group than in the CDDP group (p<0.05). Sperm motility was numerically higher in the C+R group than in the CDDP group but not statistically different (p=0.096). No significant differences in these parameters were observed between the Ctrl and RMT groups.

Histopathological examination. Figure 2A presents representative H&E-stained testicular sections from each group. H&E staining revealed marked histological abnormalities in the CDDP group, including detachment of spermatogonia from the basement membrane, decreases in spermatocyte and sperm counts, cytoplasmic vacuolation, and vascular congestion. Quantitative analysis showed that seminiferous tubule diameter was significantly smaller in the CDDP group than in the Ctrl group, supporting the presence of seminiferous tubule atrophy (p<0.001, Figure 2B). In the C+R group, these histological abnormalities were relatively mild, and seminiferous tubule diameter was significantly larger than that in the CDDP group (p<0.01).

Figure 2.

Figure 2

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Histopathological examination of testicular tissue. (A) Hematoxylin and eosin staining of tissue obtained from a single rat in each group (×400, scale bar=50 μm). (B) Quantitative analysis of seminiferous tubule diameter. (C) Immunohistochemical staining was performed on testicular tissue (×200, scale bar=100 μm). The arrowheads indicate Ki-67-positive cells in the testicular interstitium. (D) The bar chart presents the ratio of Ki-67-positive cells in the testicular interstitium. Data are presented as the mean±standard error (SE); n=8 per group. Statistical analysis was performed using one-way analysis of variance followed by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. Ctrl, Control group; CDDP, cisplatin group; RMT, ramelteon group; C+R, cisplatin plus ramelteon group.

Ki-67 immunostaining. The results of Ki-67 immunostaining of testicular tissue are presented in Figure 2C. Compared with the findings in the Ctrl and RMT groups, a significant increase in the Ki-67 positivity rate of testicular interstitial cells was observed in the CDDP group (p<0.001), whereas this rate was significantly reduced in the C+R group relative to that in the CDDP group (p<0.05, Figure 2D).

Serum testosterone. Serum testosterone levels were significantly lower in the CDDP group than in the Ctrl and RMT groups (both p<0.05, Figure 3). Meanwhile, serum testosterone levels were significantly elevated in the C+R group compared with those in the CDDP group (p<0.05).

Figure 3.

Figure 3

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Serum testosterone levels in each group. Data are presented as the mean±standard error (SE); n=8 per group. Statistical analysis was performed using the Kruskal-Wallis test followed by Steel-Dwass test. *p<0.05, **p<0.01. Ctrl, Control group; CDDP, cisplatin group; RMT, ramelteon group; C+R, cisplatin plus ramelteon group.

RT-qPCR. As presented in Figure 4, the mRNA expression of Star, Cyp11a1, Cyp17a1, and Hsd17b3 was significantly lower in the CDDP group than in the Ctrl and RMT groups (all p<0.001). Conversely, the C+R group exhibited significantly higher Hsd17b3, Cyp17a1, Cyp11a1, and Star mRNA expression than did the CDDP group (all p<0.05). Additionally, the mRNA expression of Casp3 and Il6 was significantly higher in the CDDP group than in the Ctrl, RMT, and C+R groups (all p<0.05).

Figure 4.

Figure 4

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Relative mRNA expression in testicular tissues. mRNA expression of Star (A), Cyp11a1 (B), Cyp17a1 (C), Hsd17b3 (D), Casp3 (E), and Il6 (F). Data were calculated using the ΔΔCt method and normalized to β-actin, with the values in the Ctrl group set to 1.0. Error bars indicate the mean±standard error (SE); n=8 per group. Statistical analysis was performed using one-way analysis of variance followed by Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. Ctrl, Control group; CDDP, cisplatin group; RMT, ramelteon group; C+R, cisplatin plus ramelteon group.

Discussion

Our findings support a protective role of ramelteon against acute CDDP-induced testicular dysfunction. In a rat model, ramelteon suppressed the CDDP-induced decrease in sperm counts during the acute phase. Additionally, ramelteon helped maintain the seminiferous tubule structure, improved serum testosterone levels, and partially normalized steroid synthesis-related and apoptosis- and inflammation-related gene expression.

Notably, the number of Ki-67-positive cells in the testicular interstitium was increased in the CDDP group, suggesting enhanced proliferative activity within the interstitial compartment, whereas this increase was suppressed in the C+R group. Although the involvement of other cell populations, such as inflammatory cells or fibroblasts, cannot be excluded in the absence of co-staining with cell type-specific markers, this finding suggests that ramelteon attenuated reactive changes in the testicular interstitium, which may include Leydig cell-related responses. The concomitant improvement in steroidogenesis-related gene expression and serum testosterone levels suggests that ramelteon may have mitigated interstitial and endocrine disturbances induced by CDDP.

A previous study demonstrated that ramelteon attenuated CDDP-induced injury in mouse Leydig cells in vitro (31). The present study provides complementary in vivo evidence that ramelteon mitigates acute CDDP-induced testicular injury and associated endocrine dysfunction in rats. In particular, the simultaneous attenuation of acute deterioration in sperm parameters and serum testosterone levels is important, suggesting that ramelteon may affect both seminiferous epithelial cells and Leydig cells. In fact, although significant impairments in these indicators were observed in the CDDP group, co-administration of ramelteon reduced deviations from the Ctrl group for all parameters, indicating that reproductive organ homeostasis was broadly maintained. Additionally, the suppression of CDDP-induced increases in Ki-67 positivity in the C+R group, together with the improvements in steroidogenesis-related gene expression and serum testosterone levels, suggests that ramelteon may have mitigated CDDP-induced interstitial responses and endocrine dysfunction. These findings support the possibility that the protective effects of ramelteon are not limited to seminiferous tubular morphology and sperm parameters but may also extend to Leydig cell-associated steroidogenic function.

Previously, melatonin was reported to exert protective effects in various testicular toxicity models, including toxicity induced by CDDP, heat stress, heavy metals, and chemotherapeutic agents, by activating the SIRT1-Nrf2 pathway or regulating the Akt-Bcl-2 pathway through MT1/MT2, thereby simultaneously attenuating oxidative stress, apoptosis, and inflammatory responses (31-33). However, the clinical application of melatonin has been limited by pharmacokinetic challenges, such as its short half-life and low receptor affinity (34). Conversely, ramelteon has 3- to 16-fold higher affinity for MT1/MT2 receptors than melatonin and a longer half-life (0.83-1.9 h vs. 0.75 h), enabling more sustained receptor activation (21, 22). M-II, a metabolite of ramelteon, also binds MT1/MT2 receptors and exhibits a longer elimination half-life than the parent compound, indicating a potential contribution to ramelteon’s pharmacological effects (35). Although ramelteon has been reported not to possess the free-radical scavenging function of melatonin (20), its potent signal transduction via MT1/MT2 is considered advantageous for testicular protection, and this effect might be complemented by combined treatment with other antioxidants. Recent studies have reported that ramelteon exerts protective effects against toxicity in organs such as the liver, heart muscle, and lungs (20, 23, 24), and this study extends these findings to the reproductive system.

The protective effects of ramelteon may be attributable, at least in part, to the enhancement of testicular homeostasis mechanisms through anti-apoptotic and anti-inflammatory effects mediated by MT1/MT2 receptors, similar to those observed in other organs. CDDP-induced Leydig cell damage leads to impaired steroid synthesis and subsequent reductions in testosterone secretion, whereas seminiferous tubule damage can be associated with deterioration of sperm parameters. Ramelteon suppressed these acute changes and reduced both structural and functional defects. These findings suggest that ramelteon, an approved medication, may be a pharmacologically feasible candidate for further investigation as a strategy to mitigate CDDP-induced testicular dysfunction.

This study had several limitations. First, the observation period was relatively short, and long-term effects on fertility or gonadal function, as well as outcomes such as sperm DNA damage or effects on offspring, could not be evaluated. Second, this study did not use tumor-bearing animal models, and the effect of ramelteon on the anti-tumor activity of CDDP is unclear. This point is particularly relevant because ROS-dependent apoptotic signaling contributes to CDDP-induced cytotoxicity in cancer cells (36). Therefore, future studies using tumor-bearing models are needed to determine whether ramelteon can protect normal testicular tissue without compromising the anticancer efficacy of CDDP. Third, more direct approaches, such as oxidative stress-related gene and protein expression assays and MT1/MT2 receptor inhibition experiments, are desirable to elucidate the mechanism of the observed effects. In light of these considerations, further research with a stronger emphasis on clinical utility is needed.

Conclusion

Our results demonstrated that ramelteon attenuated acute CDDP-induced testicular toxicity by maintaining seminiferous tubule morphology, suppressing apoptosis-related gene expression, reducing abnormal interstitial proliferative response, and partially restoring steroidogenesis-related gene expression. These results support the biological validity of targeting the melatonin signaling pathway to mitigate acute CDDP-induced testicular injury. Ramelteon, which has an established safety profile in humans, may represent a realistic and promising candidate for repurposing as an agent to reduce acute chemotherapy-induced testicular toxicity. Further basic research encompassing the entire spermatogenic cycle, as well as clinical studies involving patients with cancer receiving CDDP-based chemotherapy, is warranted to validate the findings of this study.

Conflicts of Interest

The Authors declare no competing financial interests or personal relationships that could be perceived to influence the work reported in this paper.

Authors’ Contributions

S. Kawamura and K. Chiba conceptualized and designed the study. S. Kawamura performed the experiments. T. Kazuki, C. Kin, Y. Yamashita, and K. Sato contributed to the experimental design and interpreted the results. S. Kawamura and K. Okada curated the data and performed the statistical analyses. S. Kawamura prepared the figures and drafted the manuscript. K. Chiba and H. Miyake reviewed and edited the manuscript. H. Miyake supervised the research. All authors reviewed the results, approved the final version of the manuscript, and agree to be accountable for all aspects of the work.

Acknowledgements

We thank Joe Barber Jr., PhD, ELS, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Artificial Intelligence (AI) Disclosure

During the preparation of this manuscript, a large language model (DeepL Translator, DeepL SE, Cologne, Germany) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.

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