Introduction
Approximately 10% to 15% of couples experience infertility, which represents one of the most significant reproductive problems [1]. Infertility can arise from various causes, with male factors accounting for 25% of cases [2]. Toxic substances, antibiotic use, radiation, pesticides, air pollution, stress, and chemotherapy drugs can all contribute to male infertility [1].
Cyclophosphamide (CYC), an alkylating cytotoxic agent with antineoplastic effects, is widely used to treat various cancers such as leukemia, multiple myeloma, lymphoma, breast and prostate cancer, sarcoma, and neuroblastoma [3,4]. Additionally, CYC exhibits immunosuppressive effects and is used in the management of autoimmune diseases and organ transplantation [5].
Even though the medication is frequently used to treat cancer, it has been associated with a number of adverse effects, including serious damage to the reproductive system [3]. The cytotoxicity of CYC is linked to azoospermia, oligospermia, germ cell apoptosis, sperm DNA double-strand breaks, and alterations in the sperm nuclear matrix composition in the epididymis and testis [1,5]. Additionally, patients receiving CYC treatment exhibit decreased testosterone levels and disrupted gonadotropin production [6].
The hepatic cytochrome P450 enzyme system produces phosphoramide mustard and acrolein, two active metabolites of CYC, although CYC itself is inert. These metabolites bind to nucleophilic sites on DNA through alkylation, resulting in cross-linking of nucleobases, which subsequently inhibits DNA replication and protein synthesis [7]. Prolonged exposure to low doses of CYC may impair fertility in male mice [8,9]. Intracellular thiol levels are critical for regulating cell susceptibility to CYC-induced damage [10]. 4-Hydroxycyclophosphamide interacts with cellular thiols to form thioalkyl derivatives, which in turn induce oxidative stress within the cell [11].
The precise mechanism underlying CYC-induced testicular toxicity remains unclear; however, numerous studies have shown that CYC can promote lipid peroxidation, generate free radicals, and trigger oxidative stress [10,12,13].
To this day, the use of natural products remains one of the most effective therapeutic strategies against CYC toxicity. Rosmarinus officinalis, commonly known as rosemary, is one such plant with needle-like leaves and purple-blue flowers [14]. Rosemary extract (RE) contains various antioxidants [15], and is used as an anti-inflammatory, antibacterial, antiviral, antifungal, antiulcerogenic, antitumor, antidepressant, and antioxidant agent [16,17]. It also boosts testosterone levels [18] and restores the ratio of seminiferous tubular cell populations [19].
Rosemary, an aromatic herb belonging to the Lamiaceae family, is widely used in the food industry as a seasoning and flavoring agent [20]. In particular, phenolic substances such as carnosol, carnosic acid, rosemanol, 7-methyl epirosmanol, isorosmanol, rosemadial, and caffeic acid are abundant in RE. These phenolics play a crucial role in the extract’s biological activities. Among them, flavonoids are particularly notable for providing strong antioxidant and anti-inflammatory effects [18]. The antioxidant capacity of rosemary leaves is further enhanced by the presence of alkaloids, tannins, glyceric acid, glycolic acid, vitamin C, many B vitamins, and choline in addition to phenolic compounds [21].
RE exerts its antioxidant activity by inhibiting lipid peroxidation, restoring oxidative balance, and neutralizing reactive oxygen species (ROS) through hydrogen donation (2-OH groups). Additionally, it scavenges harmful free radicals and activates physiological defense mechanisms [22].
The effect of RE on fertility suggests that it has strong antioxidant and anti-infertility properties, making it beneficial for reproductive health [23]. Studies indicate that RE enhances testosterone production, sperm count, and serum testosterone regulation, which positively impacts male reproductive health [24].
Chemotherapy-induced infertility can profoundly affect a person’s quality of life, so protecting germ cells and maintaining healthy testicular function during cancer treatment is especially important [25]. Thus, this study was conducted to investigate the potential protective effects of RE against CYC-induced testicular toxicity in male rats.
Methods
1. Study design
Twenty-four male Sprague-Dawley rats, weighing between 180 and 240 g, were obtained from the Comparative and Experimental Medicine Center of Birjand University of Medical Sciences in Iran. The animals were housed in groups of six under controlled environmental conditions (25±2 °C, 12-hour light/dark cycle, and normal humidity), with free access to standard food and tap water. All procedures were conducted in accordance with institutional guidelines approved by the university’s Ethical Committee for Animal Research (ethical number: IR.BUMS.REC.1403.168).
To induce testicular damage in male rats, a single intraperitoneal (i.p.) injection of CYC (200 mg/kg body weight [b.w.], i.p.) was administered [26]. For polyphenol treatment, the rats received oral RE at doses of 200 and 1,000 mg/kg. Animals (six per group) were randomly assigned to: (1) the control (CT) group (corn oil dissolved in saline, administered by gavage); (2) the CYC group (single injection of 200 mg/kg CYC b.w., i.p.); (3) CYC+RE200 (single injection of 200 mg/kg CYC b.w., i.p., plus 200 mg/kg RE for 6 weeks by gavage); and (4) CYC+RE1000 (single injection of 200 mg/kg CYC b.w., i.p., plus 1,000 mg/kg RE for 6 weeks by gavage).
2. Preparation of the extract
The dried rosemary leaves used in this experiment were purchased from a grocery market in Birjand and authenticated by a botany expert for aqueous extract preparation. To obtain the extract, 500 g of rosemary powder was mixed with 2,000 mL of distilled water and soaked at room temperature (approximately 20 to 25 °C) for 24 hours with occasional stirring. The mixture was then filtered, and the filtrate was centrifuged at 3,500 rpm for 20 minutes. The clear supernatant was concentrated to dryness using a rotary evaporator at 40 °C, yielding a powder that was stored at 4 °C until further use [27].
3. Sperm motility assessment
The epididymis was removed, promptly rinsed, and transferred to a petri dish containing 3 mL of phosphate-buffered saline (PBS). The dish was maintained at 37 °C on a hot plate to facilitate semen diffusion into the PBS solution. To ensure a uniform suspension and even distribution of spermatozoa, the diluted semen was gently agitated [28]. Sperm characteristics were evaluated according to the 2010 World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen, which outlines WHO criteria. Sperm motility was assessed under a 400× magnification microscope, with 100 to 300 sperm evaluated per animal in 10 randomly selected fields [28].
4. Testosterone level assessment
To assess testosterone levels, blood samples were obtained from each group through cardiac puncture. Serum was separated by centrifuging samples at 300 rpm for 10 minutes, after which it was stored at −20 °C until hormone analysis. Total testosterone levels were measured based on the method described by Sharma et al. [29]. Testosterone concentrations are expressed in nmol/L, and all samples were analyzed in duplicate.
5. Histopathological assessment
Each rat was anesthetized by i.p. administration of ketamine at a dose of 100 mg/kg. The testicular tissue was carefully isolated from the epididymis and adjacent structures and then immersed in 10% formalin overnight for fixation. Following fixation, the testes were sectioned for hematoxylin and eosin (H&E) staining, dried, and embedded in paraffin. To determine average seminiferous tubule diameter (100×), epithelium height (400×), and spermatogenic cell counts (1,000×), 10 images at different magnifications were randomly selected from each group. ImageJ software (National Institutes of Health) was used to analyze five randomly chosen fields. Two perpendicular diameters from the basement membrane to the epithelial border were measured to calculate mean seminiferous tubule diameter. Ten measurements were taken at different points within each tubule, from the basement membrane to the lumen, to determine average epithelial height. The ImageJ program was also used to count the numbers of spermatogonia, primary spermatocytes, spermatids, and Sertoli cells.
6. Quantitative reverse transcription polymerase chain reaction
To evaluate expression levels of the genes coding for B-cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax), and zona pellucida binding protein (ZPBP) genes, along with beta-actin (used as an internal control), quantitative reverse transcription polymerase chain reaction (qPCR) was performed across the experimental groups. Total RNA was isolated from testicular tissue using an extraction kit supplied by Pars Tous (Iran), following the manufacturer’s instructions. RNA purity was assessed using a NanoDrop device (BioTek Epoch). cDNA synthesis was then performed with a Pars Tous kit. qPCR was carried out using SYBR Green PCR Master Mix (Amplicon), and primer sequences are provided in Table 1. Amplification was conducted using an ABI StepOnePlus real-time PCR system (Applied Biosystems). Relative gene expression was determined using the comparative Ct (2−ΔΔCT) method.
7. Statistical analysis
Data were statistically analyzed using GraphPad Prism ver. 8 (GraphPad Software Inc.). Results are expressed as mean±standard deviation. Comparisons between groups were performed using one-way analysis of variance, and a p-value of less than 0.05 was considered statistically significant.
Results
1. Sperm motility
The mean percentage of sperm motility in the CYC group was significantly lower than that in the CT group (p<0.05). Conversely, sperm motility in the RE1000 group was noticeably higher than in the CYC group (p<0.05). In contrast, there was no significant difference in sperm motility between the RE-treated groups and the CT group (Table 2).
2. Testosterone levels
The results showed that serum testosterone levels in the CYC group were considerably lower than those in the CT group (p<0.01), whereas administration of RE200 did not significantly improve testosterone levels. However, testosterone levels in the RE1000 therapeutic group were significantly higher than those in the CYC group (p<0.0001). These findings suggest that RE at higher doses may exert a protective effect on testosterone levels in CYC-treated rats. Additionally, testosterone levels in the RE-treated groups did not differ significantly from the CT group (Figure 1).
3. Histopathological findings
Histopathological examination revealed that CYC treatment resulted in several adverse changes in testicular tissue, including disorganization of the germinal epithelium, detachment of spermatogenic cells, and the presence of degenerative cells (Figure 2). Seminiferous tubule diameter, epithelial height, and the quantity of Sertoli and spermatogenic lineage cells were measured for each group. Overall, the tubular diameter in the CYC group was significantly decreased compared to the CT group (p<0.01). However, co-administration of RE at both doses effectively prevented this reduction (p<0.01). Notably, the RE-treated groups exhibited tubule diameters that were not significantly different from those of the CT group, suggesting that RE preserved normal tubule structure and prevented CYC-induced damage (Figure 3).
The epithelial height was also reduced in the CYC group compared to the CT group, although the difference was not statistically significant. In contrast, animals treated with RE200 and RE1000 exhibited notably higher epithelial heights compared to the CYC group (p<0.01). These results indicate that RE, at both low and high doses, can protect epithelial structure in rats exposed to CYC. The effect appeared consistent across doses, underscoring the epithelial-supportive role of RE (Figure 4).
The findings showed that exposure to CYC led to a significant reduction in the number of spermatids and Sertoli cells compared to the CT group (p<0.0001 and p<0.05, respectively), indicating marked testicular toxicity. Both RE200 and RE1000 treatments significantly restored spermatid numbers compared to the CYC group (p<0.0001). Regarding spermatogonia, the RE1000 group exhibited more cells than the CYC group, and this difference was statistically significant (p<0.05). These results suggest that RE, particularly at higher doses, effectively protects against CYC-induced cellular loss in the seminiferous epithelium. Although the RE-treated groups displayed the highest numbers of primary spermatocytes, this increase was not statistically significant (Figure 5).
4. Quantitative real time PCR analysis
Bax expression was markedly elevated in the CYC group (p<0.05), whereas ZPBP and Bcl2 mRNA expression levels were considerably lower (p<0.0001 and p<0.001, respectively). A significant increase in Bcl2 expression was observed following treatment with both RE200 and RE1000 (p<0.0001). However, a significant upregulation of ZPBP (p<0.05) was only evident with the higher RE dose (RE1000), as RE200 did not affect ZPBP expression compared to the CYC group. Although Bax expression levels in the RE200 and RE1000 groups were lower than in the CYC group, this reduction was not statistically significant (Figure 6).
Discussion
The purpose of the current study was to investigate, using an experimental model, the possible preventive benefits of RE in reducing CYC-induced reproductive damage. To the best of our knowledge, no previous study has demonstrated a protective effect of RE against CYC-induced testicular toxicity in male rats.
CYC is a chemotherapeutic agent with potent cytotoxic and alkylating properties [30]. CYC undergoes hepatic biotransformation catalyzed by cytochrome P450 enzymes, leading to the generation of highly reactive metabolites, predominantly aldophosphamide mustard and acrolein. These metabolites are responsible for the drug’s systemic toxicity, exerting detrimental effects through the induction of oxidative stress and the overproduction of ROS and free radicals. They interact with cellular macromolecules, including DNA, membrane phospholipids, and proteins, ultimately causing significant structural and functional alterations [31].
Our results showed that CYC lowers testosterone levels, which is consistent with findings from other research [32]. These observations suggest that CYC induces downregulation of testosterone production. This is further supported by Kaya et al. [33], who reported that CYC markedly reduces hormone levels in the male reproductive system. CYC may impair the activity of key enzymes involved in testosterone synthesis, most likely through oxidative damage and altered transcriptional regulation within Leydig cells [34]. Additionally, our findings demonstrated that RE improved testicular hormone function by increasing testosterone levels. Previous studies have shown that administering RE to diabetic male rats significantly elevated testosterone levels compared to untreated controls and helped regulate testosterone secretion [19]. Heidari-Vala et al. [35] also reported that RE has androgenic potency and can boost spermatogenesis, induce testicular steroidogenesis, and scavenge free radicals.
The current investigation also revealed histological changes in the testes following CYC exposure, such as distorted and irregular seminiferous tubules and visible degradation of the spermatogenic epithelium. These structural alterations could affect both somatic and germ cell morphology and function and may result from chromosomal abnormalities or DNA damage [32,36]. Previous studies have similarly reported that CYC, as an alkylating agent, contributes to impaired spermatogenesis primarily by inducing apoptosis through elevated ROS levels and oxidative stress [37].
Furthermore, our study found that CYC treatment caused a marked decrease in both sperm count and motility. This reduction aligns with impaired spermatogenesis and a diminished number of germ cells after CYC exposure [38]. Elgazar [39] likewise reported reduced sperm numbers and defects in the spermatogenic cycle after CYC exposure. Notably, administration of RE resulted in a reduction in the percentage of abnormal sperm, accompanied by significant improvements in sperm motility and count. Because testosterone plays a key role in spermatogenesis, the elevated testosterone levels observed after RE treatment likely contributed to the improvement in sperm parameters. These findings are consistent with the results reported by Seyfi et al. [24]. One mechanism by which RE enhances sperm parameters may be its antioxidant activity. Oxidative stress impairs both enzymatic and non-enzymatic antioxidant systems in Leydig cells, which can disrupt spermatogenesis and ultimately reduce sperm count [40].
Our results also indicated that RE not only improved sperm parameters but preserved essential morphometric features such as seminiferous tubule diameter and epithelial height at values close to normal. Given its antioxidant effects, RE may act as a protective agent against CYC-induced testicular damage. Further investigation is needed to establish the efficacy of RE as a complementary antioxidant intervention during CYC-based chemotherapy.
The Bcl-2 family plays a crucial role in regulating cell death through control of the intrinsic apoptotic pathway. Bax and Bcl-2 proteins are key members of this family, and the ratio between these proteins determines whether cells survive or undergo apoptosis in response to apoptotic stimuli. Bax facilitates the release of cytochrome c from mitochondria, counteracting the protective function of Bcl-2, which inhibits cytochrome c translocation into the cytosol [41].
In animal studies, CYC treatment has been shown to increase Bax expression while decreasing Bcl-2 expression [42,43]. In our research, we similarly observed elevated Bax expression and significantly reduced Bcl-2 expression in the CYC group, aligning with previous findings.
In a mouse model of premature ovarian failure, RE, a water-soluble polyphenol, was reported to increase CYC-induced apoptosis while lowering cytochrome c and caspase-3 expression [44]. Likewise, Sahin et al. [45] showed that RE decreases Bax expression and thereby attenuates CYC-induced apoptosis in testicular tissue. Furthermore, Lin et al. [46] demonstrated that RE protects rat bone marrow mesenchymal stem cells from peroxide-induced apoptosis by reducing levels of caspase-3, caspase-9, and Bax/Bcl-2. In our study, RE appeared to reduce apoptosis in testicular tissue by increasing Bcl-2 expression.
ZPBP plays a vital role in acrosome compaction. In its absence, mice display disrupted Sertoli–spermatid junctions and fragmented acrosomes. These structural abnormalities result in malformed spermatozoa with reduced capacity to penetrate the zona pellucida. Moreover, disruption of Sertoli–spermatid junctions can impair spermatogenesis and potentially lead to azoospermia [47,48].
Our research revealed that CYC markedly reduced ZPBP gene expression, while the RE1000 group significantly elevated expression levels, although they did not fully return to control values. Although this study demonstrates the efficacy of RE in mitigating CYC-induced testicular damage, the upstream molecular mechanisms remain incompletely characterized. The observed antioxidant and anti-apoptotic effects suggest potential involvement of key signaling pathways, particularly the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response and mitogen-activated protein kinases (MAPK) signaling cascades. A limitation of this work is the absence of direct investigation into these pathways. Future studies should focus on elucidating these mechanistic aspects to provide a more comprehensive understanding of the protective actions of RE.
In conclusion, the findings suggest that RE can improve sperm motility and preserve important structural features of the testes, such as seminiferous tubule diameter and epithelial height, close to normal values. Due to its antioxidant activity, RE holds promise as a protective agent against CYC-induced testicular damage. Nonetheless, additional research is needed to fully assess its effectiveness as an antioxidant supplement during CYC chemotherapy.
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