Effects of quercetin-loaded nanoselenium on spermatogenesis in a mouse model of cyclophosphamide-induced testicular damage
Article information
Abstract
Objective
Cyclophosphamide (CP), a chemotherapeutic agent, has been shown to inhibit spermatogenesis. Accordingly, the primary objective of this study was to evaluate the potential therapeutic benefits of quercetin‑loaded nanoselenium (quercetin‑loaded selenium nanoparticles [SeNPs]) in mice treated with CP.
Methods
Thirty‑five adult male mice were randomly assigned to five groups (n=7 per group): control, quercetin‑loaded SeNPs (20 mg/kg, daily for 5 weeks), CP (200 mg/kg, single dose), treatment A (CP+quercetin‑loaded SeNPs), and treatment B (CP+quercetin, 20 mg/kg daily for 5 weeks). Sperm parameters, DNA fragmentation index, catalase activity, levels of glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde (MDA), and reactive oxygen species (ROS) were evaluated in all groups, along with histological assessments of testicular tissue.
Results
In CP‑treated mice, administration of quercetin‑loaded SeNPs (treatment A) significantly improved sperm parameters, including total count, motility, morphology, and DNA integrity. Treatment also markedly increased the numbers of spermatogonia, primary spermatocytes, spermatids, Sertoli cells, and Leydig cells in testicular tissue. Furthermore, treatment with quercetin‑loaded SeNPs resulted in a significant increase in catalase activity and GSH levels while significantly reducing GSSG, MDA, and ROS levels in CP‑induced testicular damage.
Conclusion
These findings suggest that quercetin‑loaded SeNPs enhance spermatogenesis in a CP‑induced mouse model by improving the antioxidant profile and testicular stereological parameters.
Introduction
It is well established that chemotherapeutic agents adversely affect spermatogenesis, thereby reducing male fertility potential. Such drugs impair spermatogenesis by compromising germ cell DNA integrity and inducing apoptosis in spermatogenic cells. In particular, alkylating agents are known for their genotoxic effects, frequently leading to long‑term azoospermia [1-3].
Cyclophosphamide (CP) is a widely used alkylating chemotherapeutic drug that is converted into active metabolites by liver cytochrome P450 enzymes [4,5]. CP exerts a significant negative impact on male fertility by causing DNA cross‑linking in rapidly dividing germ cells, which impairs spermatogenesis and may lead to oligozoospermia or azoospermia, potentially resulting in temporary or permanent infertility. Additionally, CP adversely affects Leydig cells, thereby reducing testosterone synthesis and causing hormonal imbalances [6-10]. Oxidative stress is a key mediator of CP’s detrimental effects on male fertility. Upon administration, CP is converted into active metabolites that increase reactive oxygen species (ROS) production. Excessive ROS inflicts oxidative damage on lipids, proteins, and DNA, particularly in the delicate germ cells involved in spermatogenesis, thus reducing sperm quality. Moreover, oxidative stress compromises the blood‑testis barrier, leading to testicular inflammation and further impairing spermatogenesis. Consequently, antioxidant supplements and pharmacological agents that enhance endogenous antioxidant defenses are under investigation to preserve male fertility during CP therapy [6-10].
Quercetin is a flavonoid with intrinsic antioxidant properties due to its capacity to scavenge free radicals and chelate heavy metals, and it is primarily found in fruits and vegetables. It exhibits various biological effects, including cardiovascular protection, anticancer, antidiabetic, and immunomodulatory actions [11,12]. Furthermore, quercetin has demonstrated potential in preventing oxidative damage to spermatogenic cells by inhibiting lipid peroxidation, preserving mitochondrial integrity, and significantly reducing germ cell apoptosis, thereby improving sperm parameters. Its therapeutic potential is being explored in dietary supplements and pharmaceutical formulations, often in combination with other antioxidants, to counteract oxidative damage and enhance testicular function [13,14].
Selenium (Se) is an essential trace element that plays critical roles in human health. As a key component of selenoproteins such as thioredoxin reductases and glutathione (GSH) peroxidases, Se helps maintain cellular redox balance. Its antioxidant properties protect cellular membranes and DNA, thus supporting overall cellular function. Se is integral to male reproductive biology and endocrine function, influencing spermatogenesis and testosterone synthesis while protecting spermatozoa from oxidative damage [15,16]. Additionally, Se supplementation has been investigated in various pathological conditions affecting male fertility, with studies demonstrating improvements in semen quality in both animal models and human subjects [17-19].
Selenium nanoparticles (SeNPs) offer a novel drug delivery system to enhance the therapeutic effects of antioxidants. SeNPs exhibit anti‑inflammatory, antioxidant, and immunomodulatory properties with significantly lower toxicity [16]. Nanoscale Se improves bioavailability, cellular uptake, and pharmacokinetic characteristics. Their multivalent surface and modifiable properties allow for efficient loading of chemical drugs or biomacromolecules via covalent or non‑covalent bonds [20]. Thus, SeNPs can function as nanocarriers for natural antioxidants, enabling controlled release and higher local concentrations of therapeutic agents, thereby effectively mitigating oxidative stress. Additionally, SeNPs possess inherent antioxidant properties, further enhancing their therapeutic potential [16,21-23]. Beyond their potential applications in male infertility [24,25], SeNPs can deliver antioxidants to protect spermatogenic cells from oxidative damage and improve sperm quality. Their dual function as both a carrier and an antioxidant makes them a promising option for treating oxidative stress‑related conditions, including chemotherapy‑induced infertility.
Based on these considerations, the present study aims to investigate the potential therapeutic effects of quercetin‑loaded SeNPs in alleviating CP‑induced testicular damage. To date, no data have addressed the impact of quercetin‑loaded SeNPs on CP‑induced testicular damage in mice.
Methods
1. Preparation of quercetin-loaded SeNPs
To prepare quercetin‑loaded SeNPs, 0.3 g of low molecular weight chitosan was dissolved in 1% acetic acid under stirring at 40 °C for 30 minutes. Separately, 0.3 g of sodium selenite was dissolved in 10 cm3 of deionized water and stirred at room temperature for 30 minutes. The sodium selenite solution was then added dropwise to the chitosan solution and allowed to react for 6 hours. At room temperature, 0.3 g of quercetin was dissolved in 10 mL of dimethyl sulfoxide (DMSO) and added dropwise to the mixture. For each milliliter of the quercetin solution, 10 µL of Tween (polysorbate) 20 was added dropwise at 15‑minute intervals. After 1 hour, 0.1 g of ascorbic acid was dissolved in 10 mL of deionized water and added dropwise. The reaction was indicated by a color change from yellow to orange. Chitosan and Tween 20 served as stabilizers. Finally, the solution was centrifuged for 30 minutes at 5,000 rpm and filtered through a 0.22‑µm syringe filter. The particle size distribution was determined by dynamic light scattering, and the zeta potential was measured using a zeta sizer.
2. Animals and experimental design
This study was approved by the Research Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.AEC.1402.082), and all procedures were conducted in accordance with relevant guidelines and regulations. Thirty‑five adult male Naval Medical Research Institute (NMRI) mice (8 weeks old, weighing 25 to 30 g) were housed at room temperature under a 12‑hour light/dark cycle with ad libitum access to food and water. The mice were randomly divided into five groups (n=7 per group): (1) control, (2) quercetin‑loaded SeNPs (20 mg/kg daily for 5 weeks), (3) CP (200 mg/kg, single intraperitoneal [ip] injection), (4) treatment A (CP+quercetin‑loaded SeNPs), and (5) treatment B (CP+quercetin, 20 mg/kg daily for 5 weeks). Mice were sacrificed using ketamine/xylazine (80 and 10 mg/kg, ip, respectively) for subsequent assessments. The CP‑induced mouse model was established based on previously published studies [9]. The study schedule is illustrated in Figure 1. Notably, on the day of sacrifice, all mice were 18 weeks old, and the CP dose was selected based on prior studies [9,26].
Comprehensive timeline of the current study. NMRI, Naval Medical Research Institute; CP, cyclophosphamide; SeNP, selenium nanoparticle; ip, intraperitoneal.
3. Semen analysis
The caudal region of the epididymis was placed in a Petri dish containing 1 mL of pre‑warmed Ham's F10 medium, minced, and incubated at 37 °C with 5% CO₂ for 15 to 20 minutes. Ten microliters of the sperm mixture were then used to evaluate sperm parameters. Sperm count, motility, and morphology were assessed using a bright‑field microscope following the previously described method [27]. Normal sperm morphology in mice was defined as having a smooth head without deformities or vacuoles, a straight, slender midpiece properly attached to the head, and a uniform tail without bends, breaks, or cytoplasmic droplets [27]. The DNA fragmentation index was determined using the sperm chromatin dispersion assay with the sperm DNA fragmentation assay kit (IVF, Tehran, Iran) according to the manufacturer's instructions, and the percentage of sperm with DNA fragmentation was recorded [28].
1) Tissue preparation and stereological study
Tissue samples were fixed in Bouin's solution for 24 hours. Serial sections of 5 and 20 μm thickness were obtained using a rotary microtome and stained with hematoxylin and eosin (H&E). Sections were selected via systematic uniform random sampling, and stereological analyses were performed in a blinded manner.
The optical dissector technique was used to estimate the numerical density (Nv) of cells in the testes [27]:
In this equation, ΣQ represents the estimated number of testicular cells. This estimation was carried out using a counting frame probe and a microcator (Heidenhain) connected to the microscope stage. The microcator was used to measure the height of the dissector (h) and the actual thickness of the tissue section (t). Additionally, ΣP denotes the total number of fields that were counted. The a/f represents the ratio of the area of the probe to the magnification, whereas the BA refers to the tissue section thickness.
4. Measurement of ROS by flow cytometry
A 100 μL sample was incubated with 2′,7′‑dichlorodihydrofluorescein diacetate (DCFH‑DA) at a concentration of 20 μM for 45 minutes at 37 °C in the dark. Subsequently, 900 μL of phosphate-buffered saline was added, and the sample was centrifuged at 1,200 rpm for 5 minutes at 4 °C. Finally, the sample was analyzed using a BD FACSLyric flow cytometer (BD Biosciences) with excitation at 495 nm.
5. Glutathione production
Eighty microliters of sample were added to test tubes, followed by 20 μL of R2 solution in each tube. The tubes were centrifuged for 10 minutes at 4,000 to 5,000 rpm. Next, 10 μL of the supernatant or standard was transferred to a microplate, and 200 μL of R3 solution was added. The mixture was then incubated at room temperature for 5 minutes before measuring absorbance at 412 nm. GSH activity was calculated based on the absorbance readings.
6. Glutathione disulfide production
Test tubes were prepared by adding 25 μL of the standard solution to some tubes and 25 μL of sample to others; an additional 25 μL of deionized water was added to the standard tubes. A working solution was then prepared by mixing 25.2 mL of glutathione disulfide (GSSG) buffer, 90 μL of cofactor, 420 μL of enzyme, 460 μL of deionized water, and 90 μL of 5,5′‑dithiobis‑(2‑nitrobenzoic acid) (DTNB). Next, 150 μL of the working solution was added to each test tube. The optical absorbance was measured at a wavelength of 412 nm (range, 400 to 420).
7. Measurement of catalase enzyme activity
Twenty microliters of the sample were added to 30 μL of catalase methanol and mixed thoroughly. Then, 20 μL of the catalase substrate was added to each well to initiate the reaction. The plate was covered and incubated at room temperature for 20 minutes. Subsequently, 30 μL of the catalase stop solution was added to each well, followed by 30 μL of catalase chromogen, and incubated for 10 minutes at room temperature. Finally, 10 μL of catalase periodate was added, and after a further 5 minutes, absorbance was measured at a wavelength range of 520 to 560 nm.
8. Evaluation of lipid peroxidation (malondialdehyde levels)
After preparing the necessary solutions and allowing them to reach ambient temperature, 50 μL of each sample or standard solution was added to separate microtubes. Subsequently, 1 mL of chromogenic solution was added, and the tubes were incubated for 1 hour. The tubes were then placed in boiling water, cooled on ice, and centrifuged for 10 minutes at 4,000 rpm. Next, 200 μL of the pink supernatant was transferred to a microplate, and absorbance was measured at 535 nm using an enzyme-linked immunosorbent assay reader. Malondialdehyde (MDA) levels were calculated based on the standard curve obtained.
9. Statistical analysis
Data were analyzed using GraphPad Prism ver. 9 (GraphPad Software Inc.) and are presented as mean±standard deviation. The Shapiro–Wilk test was employed to assess normality. Statistical significance was determined by one‑way analysis of variance (ANOVA) followed by Tukey’s post hoc test, with p<0.05 considered statistically significant. All significant differences are reported at p<0.05 for clarity.
Results
1. Characterization of quercetin-loaded SeNPs
As shown in Figure 2, the quercetin‑loaded SeNPs had a particle size of 108 nm and a zeta potential of –5.8 mV.
Characterization of the quercetin-loaded selenium nanoparticles (SeNPs). (A) Zeta potential analysis of quercetin-loaded SeNPs. The surface charge was determined to be –5.8 mV. Measurements were performed using a Zetasizer (Malvern Panalytical). (B) Size distribution profile of quercetin-loaded SeNPs. The average diameter was measured as 108 nm by dynamic light scattering (DLS).
2. Sperm parameters
As depicted in Figure 3, CP administration significantly reduced sperm count, total motility, progressive motility, normal morphology, and DNA integrity compared to controls (p<0.05). Treatment with quercetin‑loaded SeNPs (treatment A) significantly improved sperm count, total motility, progressive motility, and DNA integrity relative to the CP group (p<0.05); however, it did not significantly affect the percentage of normal sperm morphology (p>0.05). In contrast, treatment B (CP+quercetin) did not yield significant improvements in any of these parameters compared to the CP group (p>0.05). No significant differences were observed among the study groups regarding sperm non‑progressive motility (p>0.05). Additionally, there were no significant differences between the control and quercetin‑loaded SeNPs groups (p>0.05) for these parameters.
Comparison of sperm parameters between study groups. (A-F) All data are presented as mean±standard deviation. Normal sperm morphology (E) was assessed based on the following criteria: a smooth head without deformities or vacuoles, a straight and slender midpiece properly attached to the head, and a uniform tail free of bends or breaks, with no cytoplasmic droplets or abnormal shapes. (G, H) Representative photomicrographs of sperm with intact (G) and fragmented (H) DNA content (×100 objective). a, fast progressive motility; b, slow progressive motility; a+b, progressive motility; c, non-progressive motility; SeNP, selenium nanoparticle. a),b),c)No statistically significant differences between groups (p>0.05), whereas different letters indicate significant differences (p<0.05).
3. Number of cells in testicular tissue
Figure 4 displays histological sections of testicular tissue from the study groups, highlighting CP‑induced effects on the seminiferous tubules’ germinal epithelium. Figure 5A-5E presents the counts of various cell types in the testicular tissue, while Figure 5F shows a photomicrograph of mouse testicular tissue illustrating different cell types.
Representative photomicrographs of hematoxylin and eosin-stained seminiferous tubules across study groups (×10 and ×40 objective). Microscopic examination of the testicular tissue structure in the cyclophosphamide (CP) group revealed significant damage to the germinal epithelium, shrinkage of the seminiferous tubule walls, and an increased intertubular space compared to the control group. Furthermore, both treatment groups showed a marked improvement in the testicular tissue structure compared to the CP group. Treatment A, CP+quercetin-loaded selenium nanoparticles (SeNPs); treatment B, CP+quercetin.
Quantification of testicular cell types across study groups. (A-E) Data are expressed as mean±standard deviation. (F) Representative photomicrographs of hematoxylin and eosin-stained testicular tissue illustrating distinct cell types. Treatment A, cyclophosphamide (CP)+quercetin-loaded selenium nanoparticles (SeNPs); treatment B, CP+quercetin; SeNP, selenium nanoparticle; LC, Leydig cell; SC, Sertoli cell; SG, spermatogonia; PS, primary spermatocyte; ST, spermatid. a),b),c)No statistically significant differences between groups (p>0.05), whereas different letters indicate significant differences (p<0.05).
CP injection significantly reduced the numbers of spermatogonia, primary spermatocytes, spermatids, Leydig cells, and Sertoli cells compared to the control group (p<0.05). Both treatment A (CP+quercetin‑loaded SeNPs) and treatment B (CP+quercetin) groups exhibited significant increases in spermatogonia, spermatids, Leydig cells, and Sertoli cells relative to the CP group (p<0.05). However, only treatment A showed a significant increase in primary spermatocyte numbers compared to the CP group (p<0.05), whereas the increase in the treatment B group was not significant (p>0.05). No significant differences were found between the control and quercetin‑loaded SeNPs groups for these parameters (p>0.05).
4. Catalase activity and markers of oxidative stress
Figure 6 illustrates the results for catalase activity and oxidative stress markers across the study groups. The CP group exhibited significant reductions in catalase activity and GSH levels, along with significant increases in GSSG, MDA, and ROS levels in testicular tissue compared to controls (p<0.05). Both treatment A and treatment B groups showed significant improvements in catalase activity and GSH levels, as well as significant reductions in GSSG, MDA, and ROS levels relative to the CP group (p<0.05). Notably, treatment A was significantly more effective than treatment B in enhancing the antioxidant/oxidant profile in CP‑induced mice, as evidenced by higher catalase activity and GSH levels and lower GSSG and ROS levels (p<0.05). Additionally, no significant differences were observed between the control and quercetin‑loaded SeNPs groups (p>0.05).
Catalase activity and markers of oxidative stress in the study groups. (A-E) All data are presented as mean±standard deviation. SeNP, selenium nanoparticle; Treatment A, cyclophosphamide (CP)+quercetin-loaded selenium nanoparticles (SeNPs); treatment B, CP+quercetin; GSH, glutathione; GSSG, glutathione disulfide; MDA, malondialdehyde; ROS, reactive oxygen species. a),b),c)No statistically significant differences between groups (p>0.05), whereas different letters indicate significant differences (p<0.05).
Discussion
This study investigated the therapeutic efficacy of quercetin‑loaded SeNPs in a mouse model of CP‑induced testicular damage, providing insights into potential mechanisms, therapeutic implications, and future research directions. To our knowledge, no previous studies have examined the protective effects of quercetin‑loaded SeNPs against CP‑induced testicular damage.
CP treatment caused a significant decline in sperm parameters, whereas quercetin‑loaded SeNPs demonstrated promising efficacy in improving sperm count, motility (both total and progressive), morphology, and DNA integrity in CP‑treated mice. Furthermore, treatment with quercetin‑loaded SeNPs significantly increased the number of testicular cells, indicating its potential to mitigate chemotherapy‑induced testicular damage. The observed increase in Sertoli cell numbers is unlikely due to de novo proliferation, given that adult Sertoli cells are postmitotic [29]; rather, it likely reflects a protective effect by reducing CP‑induced apoptosis or structural damage and preserving the existing Sertoli cell population. Additionally, these treatments may enhance the testicular microenvironment, thereby supporting Sertoli cell survival and functional recovery—mechanisms that warrant further investigation. Previous studies have shown that Se supplementation can improve spermatogenesis in CP‑induced mouse models, partly by upregulating glutathione peroxidase 4 (GPx4), an enzyme that protects against membrane lipid peroxidation, and by increasing the expression of solute carrier family 7 member 11 (SLC7A11), which is essential for cysteine/GSH homeostasis in Sertoli cells [30,31]. Moreover, quercetin has been reported to exert preventive effects against CP‑induced testicular damage by modulating multiple pathways involved in apoptosis and oxidative stress responses [32]. However, none of these studies employed a nanoparticle‑based approach to combine these effects, as was achieved with quercetin‑loaded SeNPs.
In this study, CP administration significantly reduced catalase activity and GSH levels while markedly increasing GSSG (oxidized GSH), MDA, and ROS in mouse testicular tissue. These findings support other studies indicating that oxidative stress is a key contributor to CP‑induced testicular injury [33,34]. Treatment with both quercetin‑loaded SeNPs and quercetin significantly increased catalase activity and GSH levels and decreased GSSG, MDA, and ROS compared to the CP group. Notably, quercetin‑loaded SeNPs were significantly more effective than quercetin alone in improving the antioxidant/oxidant profile, underscoring the promise of a nanotechnology approach that combines the antioxidant properties of both quercetin and Se. quercetin has been shown to protect spermatogenic cells by preventing lipid peroxidation, preserving mitochondrial integrity, and reducing germ cell apoptosis, thereby improving sperm parameters [13,14]. As mentioned earlier, Se, a critical component of selenoproteins such as GPxs and thioredoxin reductases, maintains cellular redox equilibrium and protects cellular membranes and DNA [15,16]. Thus, the combined delivery of quercetin and Se via quercetin‑loaded SeNPs may potentiate the antioxidant effect compared to quercetin alone.
In conclusion, our findings indicate that quercetin‑loaded SeNPs enhance spermatogenesis in a CP‑induced mouse model by improving both the antioxidant profile and testicular stereological parameters. Moreover, quercetin‑loaded SeNPs outperform quercetin alone in mitigating oxidative stress, a key factor in chemotherapy‑induced testicular dysfunction. Further studies are necessary to thoroughly evaluate the safety, therapeutic efficacy, and optimal dosing of quercetin‑loaded SeNPs, which will be crucial in establishing their translational potential for future clinical applications in combating chemotherapy‑induced reproductive toxicity.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgements
This study is derived from the thesis of Mahsa Ghaffari Novin, a Ph.D. student of reproductive biology at the Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Author contributions
Conceptualization: MGN (Mahsa Ghaffari Novin), MGN (Marefat Ghaffari Novin), HN, ZSM. Methodology: MGN (Mahsa Ghaffari Novin), MGN (Marefat Ghaffari Novin), MAA, PR, AM, HM, ZSM. Formal analysis: HN, ZSM. Data curation: HN, ZSM. Funding acquisition: HN, ZSM. Project administration: HN, ZSM. Visualization: HN, ZSM. Validation: MGN (Mahsa Ghaffari Novin), HN, ZSM. Investigation: MGN (Mahsa Ghaffari Novin), MGN (Marefat Ghaffari Novin), MAA, PR, AM, HM, ZSM. Writing-original draft: MGN (Mahsa Ghaffari Novin), MGN (Marefat Ghaffari Novin), AM, HM. Writing-review & editing: PR, HN, ZSM. Approval of final manuscript: MGN (Mahsa Ghaffari Novin), MGN (Marefat Ghaffari Novin), MAA, PR, AM, HM, HN, ZSM.