Prepubertal co-exposure to nandrolone and royal jelly alters testicular histological structure and spermatogenic dynamics in male mice: Insights from histomorphometry
Article information
Abstract
Objective
Nandrolone, a synthetic anabolic steroid, is frequently used for muscle enhancement but is known to compromise male fertility, particularly when administered at supraphysiological doses. Conversely, royal jelly exhibits antioxidant activity and may exert protective effects on reproductive function. This study aimed to assess the histomorphological effects of nandrolone, royal jelly, and their concurrent administration on testicular architecture during pubertal development.
Methods
Thirty-five male Syrian mice (4–5 weeks old, approximately 25 g) were randomly assigned to five groups (n=7): untreated control, sesame oil vehicle, nandrolone (10 mg/kg/week), royal jelly (500 mg/kg/day), and combined nandrolone plus royal jelly. On day 32, animals were euthanized, and testicular tissues were harvested for morphological and histological evaluation. Paraffin-embedded sections were stained with hematoxylin and eosin, and seminiferous tubule diameters were quantitatively assessed.
Results
Nandrolone administration increased testicular weight and the gonadosomatic index relative to controls. Royal jelly similarly enhanced testicular metrics. However, co-treatment markedly reduced testicular weight, gonadosomatic index, and lumen diameter, while germinal epithelium thickness was increased.
Conclusion
Nandrolone alone may not impair fertility during pubertal maturation, and royal jelly may enhance reproductive potential when administered prepubertally. Nonetheless, their combined administration appears to disrupt spermatogenic architecture, potentially impeding sperm transit through epithelial hyperplasia and delaying testicular maturation.
Introduction
Reproductive health is critically dependent on the structural and functional integrity of the testes, which regulate spermatogenesis and androgen production. Disruption of testicular architecture, whether arising from genetic, environmental, or pharmacological factors, can result in infertility with significant clinical implications [1-6].
Nandrolone decanoate, a synthetic anabolic steroid, is widely misused among adolescents and athletes. Its administration suppresses gonadotropin secretion and impairs testicular function, leading to reduced sperm count, altered sperm morphology, and hormonal imbalance. Although partial recovery has been reported following cessation, the long-term consequences of exposure during prepubertal development remain poorly understood [7-10].
Royal jelly, a bioactive secretion produced by honeybees, has demonstrated antioxidant and fertility-enhancing properties in experimental models. While low doses may improve testicular parameters, higher doses have been associated with hormonal dysregulation [11]. This study investigated the histological effects of nandrolone exposure during the prepubertal period in male mice and evaluated whether concurrent administration of royal jelly could mitigate or reverse testicular alterations. By focusing on histomorphometric outcomes, this study aimed to clarify the risks associated with adolescent steroid misuse and to explore the potential protective role of royal jelly [11,12]. Hormonal and immunohistochemical analyses are planned for future investigations.
Methods
Thirty-five male Syrian mice (4–5 weeks old, approximately 25 g) were obtained from the Faculty of Pharmacy, Ferdowsi University of Mashhad, and housed under standard laboratory conditions (20±2 °C, 12-hour light/dark cycle, 30%–40% humidity). Animals had ad libitum access to food and water, and all experimental procedures adhered to international ethical guidelines. Ethical approval for this study was obtained from the Institutional Animal Care and Use Committee at Ferdowsi University of Mashhad prior to initiation of the experiments (Project Code: 38386). All animals were screened for general health before inclusion, and no animals were excluded based on baseline characteristics.
Nandrolone decanoate (Iran Hormone Co.) was diluted to a final dose of 10 mg/kg using sesame oil as the vehicle. Group allocation was randomized using a computer-generated sequence. Histological assessments and statistical analyses were performed by investigators blinded to treatment groups. Mice were randomly assigned to five experimental groups (n=7) and treated for a total duration of 32 days: group 1: control (no treatment); group 2: vehicle control (weekly sesame oil injection); group 3: nandrolone (10 mg/kg intramuscular injection, once weekly); group 4: royal jelly (500 mg/kg oral gavage, daily); and group 5: combined nandrolone+royal jelly (same doses as above).
The selected doses were based on previous studies evaluating testicular toxicity and antioxidant efficacy [9,13]. Following completion of the treatment period, mice were euthanized, and the left testes were excised and weighed. Morphometric parameters, including testicular length, width, and diameter, were recorded. The gonadosomatic index was calculated using the following formula: gonadosomatic index (%)=(testis weight/final body weight)×100.
Testes were fixed in Bouin’s solution, dehydrated, embedded in paraffin, and sectioned at a thickness of 5 µm. Sections obtained from the central region of the testis were stained with hematoxylin and eosin (H&E) for histological analysis [14]. Nandrolone was administered via intramuscular injection, and royal jelly was administered by oral gavage, following standardized protocols (Figure 1) [7,13,15]. The histological evaluation aimed to assess structural alterations in the testes and seminiferous tubules following treatment. Bouin’s fixative was prepared by mixing 75 mL saturated picric acid, 25 mL formalin (37%), and 5 mL glacial acetic acid to ensure optimal nuclear preservation. Paraffin-embedded sections were obtained from the central testicular region and cut at 5 µm using a rotary microtome. For each testis, three non-overlapping sections from the central region were analyzed to ensure representative sampling. Sections were stained with H&E to visualize seminiferous tubule morphology, lumen diameter, and germinal epithelium thickness [16].
The standardized protocol for administering intramuscular injections and oral gavage in mice. Proper restraint techniques were employed to minimize stress and ensure consistent delivery of agents across all treatment groups. This procedural consistency was essential for maintaining experimental reliability.
Morphometric measurements were performed at 10× magnification using ImageJ software (National Institutes of Health) (Figure 2) [17], and mean values derived from three independent measurements were used for analysis. Data were analyzed using GraphPad Prism 8.2.1 (GraphPad Software, Inc., San Diego, CA, USA) with parametric statistical methods. Tukey’s post hoc test was applied for intergroup comparisons, and the Student’s t-test was used for pairwise mean analyses. Graphs were generated using Microsoft Excel, and statistical significance was set at p<0.05. Table 1 summarizes changes in testicular weight, length, width, and diameter across all groups. While the Syrian mouse model provides valuable insights into testicular histology, extrapolation to human physiology should be approached with caution.
The calibration and measurement workflow used for morphometric analysis. A transparent grid overlay was applied to histological sections to quantify seminiferous tubule diameter, lumen size, and germinal epithelium thickness. Color-coded reference lines (yellow, red, and blue) facilitated precise measurement of structural parameters, while perimeter and cross-sectional area were calculated using digital contour tracing. Sections were stained with hematoxylin and eosin (H&E) for histological analysis.
Mean values of testicular morphometric parameters, including weight, length, diameter, and width, recorded at the time of tissue harvesting
Exact p-values were reported wherever raw statistical outputs were available. For figures derived from earlier datasets in which raw outputs were no longer accessible due to archival constraints, conventional threshold notation (e.g., p<0.001) was retained based on the original analyses.
Results
This study aimed to assess the combined impact of nandrolone and royal jelly on testicular histomorphology and spermatogenic indices during the prepubertal phase in male Syrian mice. Thirty-five animals (4–5 weeks old, approximately 25 g) were randomly assigned to five groups and treated over a 32-day experimental period. Nandrolone was administered intramuscularly at 10 mg/kg/week [9,10], and royal jelly was delivered via oral gavage at 500 mg/kg/day [11]. Sesame oil served as the vehicle control.
Significant reductions in lumen diameter were observed in the nandrolone–royal jelly group compared with the control (p<0.001), nandrolone (p<0.001), and royal jelly groups (p<0.001) (Figure 3). The nandrolone group also exhibited a significant decrease relative to the sesame oil group (p<0.001), indicating that both nandrolone alone and its combination with royal jelly adversely affected luminal patency (Figure 4).
Lumen diameter was significantly reduced in the nandrolone (ND)–royal jelly (RJ) group compared with all other groups (p<0.001), including ND and RJ monotherapies. These reductions suggest that concurrent exposure may intensify epithelial proliferation and obstruct sperm passage. The ND group also exhibited a significant reduction relative to the sesame oil (SS) group (p<0.001), reinforcing its suppressive effect on luminal patency. Con, control. a),b)Statistically significant.
Morphological observations indicating that sesame oil (SS) treatment (10 mg/kg) resulted in a mild increase in lumen diameter, which may support fertility. In contrast, co-administration of nandrolone (ND) and royal jelly (RJ) produced near-complete luminal occlusion, suggesting reduced reproductive capacity. Sections were stained with hematoxylin and eosin (H&E) for histological analysis.
The royal jelly group demonstrated a significant increase in seminiferous tubule diameter compared with the nandrolone group (p<0.05), suggesting a protective or stimulatory effect on tubular expansion. However, no statistically significant differences were detected in tubule area or cross-sectional area across treatment groups (p>0.05) (Figures 4-6).
While the overall analysis of variance indicated no statistically significant differences in tubule perimeter across all groups (p>0.05), a modest, statistically significant increase was detected upon pairwise comparison in the royal jelly (RJ) group relative to the nandrolone (ND)+RJ group (p<0.05), possibly reflecting partial preservation of tubular integrity under RJ monotherapy. Con, control; SS, sesame oil (sham). a)Statistically significant.
Cross-sectional area measurements of seminiferous tubules did not differ significantly across treatment groups (p>0.05), indicating that observed lumen narrowing was primarily attributable to epithelial thickening rather than overall tubule shrinkage. Con, control; SS, sesame oil; ND, nandrolone; RJ, royal jelly.
Germinal epithelium thickness was markedly elevated in the nandrolone–royal jelly group, resulting in near-complete luminal occlusion (p<0.001) (Figure 7). Although overall changes in the tubular microenvironment were not statistically significant (p>0.05) (Figure 5), subtle structural differences were observed between the royal jelly and nandrolone–royal jelly groups, suggesting potential epithelial remodeling.
Quantitative assessment of germinal epithelium thickness demonstrated that nandrolone (ND) alone did not significantly alter epithelial structure (p>0.05). In contrast, its combination with royal jelly (RJ) resulted in marked epithelial hyperplasia and luminal narrowing (p<0.001). The RJ+ND group exhibited significantly greater germinal layer thickness compared with both RJ and ND groups, suggesting a synergistic proliferative response that may impair spermatogenic flow. Con, control; SS, sesame oil (sham). a)Statistically significant.
Morphological assessment revealed a significant increase in testicular weight in the nandrolone group compared with controls. The royal jelly group also exhibited increased testicular mass, whereas the nandrolone–royal jelly group showed a notable reduction relative to both monotherapy groups. Gonadosomatic index analysis confirmed these patterns, with statistically significant differences observed in the nandrolone and nandrolone–royal jelly groups compared with controls.
Initial body weight was recorded using a digital scale with a precision of 0.001 g, yielding a mean value of 25.378±6.875 g. No statistically significant differences in body weight were observed among groups on day 32 (p>0.05). Although fat and muscle distribution were not quantitatively assessed, prior studies suggest that nandrolone may increase lean muscle mass [18,19], while sesame oil exerts minimal physiological impact.
On day 32, histological analysis revealed distinct intergroup differences in tubule diameter (Figure 8), lumen diameter (Figure 3), germinal epithelium thickness (Figure 7), and tubule area (Figure 5). In the sesame oil group, a slight increase in lumen diameter was observed, suggesting a mild positive influence on fertility. In contrast, the nandrolone–royal jelly group exhibited near-complete luminal occlusion, indicative of reduced reproductive potential.
Seminiferous tubule diameter was significantly greater in the royal jelly (RJ) group than in the nandrolone (ND) group (p<0.05), suggesting a trophic or regenerative effect. In contrast, nandrolone treatment reduced tubule diameter, consistent with its known suppressive impact on testicular architecture. Con, control; SS, sesame oil (sham). a)Statistically significant.
Nandrolone monotherapy did not significantly alter germinal epithelium thickness compared with controls (p>0.05). However, co-administration with royal jelly (10 and 500 mg/kg) significantly increased epithelial thickness (p<0.001), leading to pronounced lumen narrowing. Comparative analysis demonstrated highly significant differences between the nandrolone and nandrolone–royal jelly groups, with the latter exhibiting greater epithelial expansion and compromised lumen integrity.
Discussion
The selected dose of nandrolone (10 mg/kg) was based on prior studies demonstrating testicular toxicity and impaired spermatogenesis [9]. Royal jelly has been reported to improve male reproductive function, particularly spermatogenic activity and hormonal balance, at doses ranging from 200 to 500 mg/kg [13]. In the present study, royal jelly monotherapy exerted beneficial effects on testicular morphology [20], whereas its co-administration with nandrolone resulted in reduced testicular weight and narrowed lumen diameter, potentially mediated by increased oxidative stress or synergistic cytotoxicity [21].
Histological evaluation revealed a non-significant increase in lumen diameter in the sesame oil group compared with the nandrolone group. Nandrolone (10 mg/kg) appeared to stimulate germinal cell proliferation, as indicated by increased epithelial thickness [22]; however, it also produced a more pronounced reduction in lumen diameter than sesame oil.
The nandrolone–royal jelly group exhibited a significant elevation in germinal epithelium thickness, accompanied by marked luminal constriction and near-complete occlusion (p<0.001). Compared with either agent administered alone, the combined treatment induced more severe narrowing, suggesting excessive germinal cell proliferation that may impede sperm transit. This phenomenon may reflect heightened mitotic activity or dysregulated cell-cycle progression within the seminiferous epithelium [23].
Significant intergroup differences were observed in testicular length, diameter, and width. The royal jelly group demonstrated the most substantial increase in testicular length compared with all other groups (p<0.001). Similarly, testicular diameter was significantly greater in the royal jelly group than in the control, sesame oil, and nandrolone groups (p<0.05). Although the nandrolone–royal jelly group showed a modest increase in diameter, this value remained notably lower than that observed in the royal jelly group alone.
With respect to testicular width, the royal jelly group again exhibited the most pronounced enhancement, followed by the nandrolone–royal jelly group. Statistically significant differences were detected between the royal jelly group and all other groups (p<0.001), reinforcing its positive influence on testicular growth. However, co-administration with nandrolone attenuated this effect, suggesting a potential antagonistic interaction between the two agents.
Overall, royal jelly improved testicular dimensions, whereas nandrolone exerted suppressive effects on these parameters. Their combination resulted in a net reduction in testicular size relative to control values, indicating that concurrent administration may negate individual benefits. Interestingly, sesame oil, which was used as the vehicle for nandrolone, elicited effects similar to those observed with nandrolone itself, implying that it may not be entirely biologically inert.
Royal jelly alone increased germinal epithelium thickness, and its combination with nandrolone further amplified this effect, potentially reflecting an additive influence on spermatogenic cell regeneration [20,24,25]. However, neither sesame oil nor nandrolone alone produced statistically significant changes in epithelial thickness.
The combined treatment administered during the prepubertal window appeared to exert deleterious effects on spermatogenesis, characterized by excessive proliferation of seminiferous epithelial cells. Such hyperproliferation may disrupt normal cell-cycle regulation and ultimately compromise fertility potential.
Further comparison of seminiferous tubule dimensions revealed that nandrolone significantly reduced tubule diameter relative to sesame oil, whereas sesame oil itself did not significantly deviate from baseline values. No statistically significant differences in tubule diameter were observed between the combined treatment group and the individual nandrolone or royal jelly groups when each was compared with controls.
Analysis of the tubular microenvironment revealed a notable distinction only between the royal jelly and nandrolone–royal jelly groups, with royal jelly alone being associated with a more favorable histological profile. Tubule area measurements did not differ significantly across groups, suggesting that lumen narrowing was primarily attributable to epithelial thickening rather than to overall tubule expansion.
Based on the collected data, the reduction in seminiferous tubule lumen diameter observed in the nandrolone–royal jelly group was not attributable to a decrease in overall tubule size, but rather to abnormal thickening of the germinal epithelium. Although high-dose nandrolone has previously been associated with seminiferous damage, the present findings indicate that such effects were not uniformly manifested in this experimental model. Interestingly, sesame oil exhibited mild beneficial effects, potentially related to its nutritional constituents. Furthermore, evidence from previous studies suggests that sesame oil does not exert significant biological effects that would interfere with or synergize with the action of nandrolone. Therefore, sesame oil can be considered an appropriate vehicle for steroid administration without substantially confounding experimental outcomes.
Royal jelly at a dose of 500 mg/kg, when administered during the prepubertal stage, appeared non-toxic and supportive of testicular health when used alone. However, its combination with nandrolone resulted in near-complete luminal occlusion, which is likely to impair sperm transport. These findings suggest a high probability of infertility when both agents are administered concurrently during critical developmental windows.
Significant morphological differences were observed among groups, particularly with respect to testicular length, diameter, and width. The royal jelly group demonstrated the most pronounced increases across all parameters (p<0.001 for length and width; p<0.05 for diameter). Although the nandrolone–royal jelly group exhibited some enhancement, values remained significantly lower than those observed with royal jelly alone, indicating a potential antagonistic interaction.
Overall, royal jelly improved testicular dimensions, whereas nandrolone exerted suppressive effects. Their combined administration resulted in reduced testicular size relative to controls, suggesting that concurrent exposure may negate individual benefits. Notably, sesame oil, which was used as the vehicle for nandrolone, produced effects similar to those observed with nandrolone, implying that it may not be entirely biologically inert.
Histological analysis revealed a greater lumen diameter in the sesame oil group than in the nandrolone group, although this difference did not reach statistical significance. Nandrolone (10 mg/kg) appeared to stimulate germinal cell proliferation, as evidenced by increased epithelial thickness, while also producing a more pronounced reduction in lumen diameter.
The nandrolone–royal jelly group exhibited significant epithelial thickening accompanied by near-complete luminal occlusion (p<0.001). Compared with monotherapy groups, the combined treatment induced more severe narrowing, likely driven by excessive germinal cell proliferation, potentially reflecting heightened mitotic activity or disrupted cell-cycle regulation. Royal jelly alone increased germinal layer thickness, and its combination with nandrolone further amplified this effect, suggesting a possible additive influence on spermatogenic cell regeneration. However, neither sesame oil nor nandrolone alone significantly altered epithelial thickness.
The combined treatment administered during the prepubertal phase exerted an unfavorable impact on spermatogenesis, characterized by hyperproliferation of seminiferous epithelial cells. Such activity may interfere with normal cell-cycle progression and compromise fertility. Further comparison demonstrated that nandrolone significantly reduced tubule diameter relative to sesame oil, whereas sesame oil itself showed no deviation from baseline. No significant differences in tubule diameter were observed between the combined group and individual treatment groups. Tubular microenvironment analysis revealed a significant difference only between the royal jelly and nandrolone–royal jelly groups, with royal jelly alone associated with a more favorable histological profile. Despite this, tubule area measurements did not differ significantly, indicating that lumen narrowing was primarily attributable to epithelial thickening rather than overall tubule expansion.
In summary, while royal jelly may enhance testicular structure and function, its concurrent use with nandrolone during prepuberty may exacerbate germinal cell proliferation to a pathological extent. The resulting disruption of lumen integrity and microarchitecture suggests a high likelihood of impaired spermatogenesis and infertility under combined exposure. These findings underscore the potential risks of combining anabolic steroids with bioactive supplements during sensitive developmental periods. Royal jelly may potentiate germinal cell proliferation when co-administered with nandrolone, potentially obstructing sperm passage. Therefore, simultaneous use of these agents, particularly at high doses, is strongly discouraged due to their antagonistic interaction and detrimental impact on reproductive health.
It is important to note that the present study was limited to histological evaluations. To achieve a more comprehensive understanding of testicular function and reproductive outcomes, future investigations will incorporate hormonal profiling and immunohistochemical analyses. These follow-up studies are currently being designed as part of an extended research project aimed at enhancing the clinical relevance and translational value of the findings.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
The author would like to express his sincere gratitude to Associate Professor Roya Lari from Ferdowsi University of Mashhad, Professor Nasser Mahdavi Shahri from Kavian Institute of Higher Education, for their invaluable guidance and support during the preparation of this manuscript.
Author contributions
Conceptualization: AY. Methodology: AY. Formal analysis: AY. Data curation: AY. Supervision: AY. Writing-original draft: AY, RL, NMS. Writing-review & editing: AY, RL, NMS. Approval of final manuscript: AY.