Introduction
Infertility is a significant global health concern, affecting approximately 15% of couples worldwide, with male factors contributing to nearly half of all cases [1]. In mammals, a reduced testicular temperature is essential for normal spermatogenesis; accordingly, testicular temperature is typically 4–5 °C lower than core body temperature [2]. However, testicular temperature can increase due to several factors, including cryptorchidism, varicocele, and lifestyle-related exposures such as frequent use of hot baths or saunas, clothing choices, and prolonged cycling. These increases can ultimately lead to damage to germ cells, somatic cells, and sperm parameters [3].
Both transient and chronic scrotal hyperthermia can activate signaling pathways associated with cell death in testicular tissue. Tumor necrosis factor-alpha (TNF-α) is a multifunctional cytokine involved in the regulation of immune responses and apoptosis. TNF-α activates pathways in the testes that promote germ cell apoptosis. These pro-inflammatory mediators affect Sertoli and Leydig cells, which express tumor necrosis factor receptor 1 (TNFR1). Binding of TNF-α to TNFR1 activates nuclear factor kappa B (NF-κB) signaling in Sertoli cells, leading to increased expression of Fas ligand (FASL) and subsequent apoptosis of germ cells expressing Fas [4-6].
Medicinal plants have long been used as alternative therapeutic agents and are often perceived as safer than synthetic drugs. The use of medicinal plants dates back to prehistoric times, with diverse cultures employing them to treat various diseases [7]. Nigella sativa (black seed), a dicotyledonous plant belonging to the Ranunculaceae family, has a long-standing historical and cultural significance [8]. It is widely distributed across Southern Europe, North Africa, and Asia Minor and has been reported to improve several health conditions, including infertility [9].
Black seed contains multiple bioactive compounds, including thymoquinone, terpenes, saponins, flavonoids, and alkaloids, which contribute to its diverse therapeutic effects. These include antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. Germ cells are particularly susceptible to oxidative stress, which can impair their proliferation and differentiation [10]. Previous studies have demonstrated that the antioxidant properties of black seed may make it a promising candidate for improving male fertility [11].
However, the antioxidant and anti-inflammatory effects of black seed have not been fully investigated in models of heat-induced testicular stress. Therefore, this study aimed to evaluate the effects of black seed on antioxidant and anti-inflammatory activity in the testicular tissue of adult male mice subjected to transient scrotal hyperthermia.
Methods
1. Animals
Twenty-four 7-week-old male Naval Medical Research Institute (NMRI) mice weighing 25–30 g were purchased from the Pasteur Institute of Iran. To allow acclimatization to the animal facility conditions, the animals were housed in metal cages for 1 week at room temperature (22–24 °C) under a 12-hour light/dark cycle, with standard chow and water available ad libitum. All study protocols were reviewed and approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.AEC.1404.063).
2. Experimental design
Twenty-four male mice were randomly divided into the following groups:
Group I (Control): Animals in this group received normal saline (0.9%) orally once daily for 35 days (n=8).
Group II (Hyperthermia): Animals in this group were subjected to transient scrotal hyperthermia five times on alternate days (n=8) [12].
Group III (hyperthermia+black seed [Hyper+BS]): Following induction of hyperthermia, animals received black seed at a dose of 300 mg/kg orally once daily for 35 days (n=8) [13].
3. Transient scrotal hyperthermia model
In the hyperthermia group, the animals’ testes were exposed to water at 43 °C for 20 minutes, five times on alternate days. The mice were first anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). Their lower bodies, including the scrotum and hind limbs, were then immersed in a water bath, after which they were dried and returned to their cages [12].
4. Preparation of black seed powder
N. sativa seeds were purchased from a reputable supplier and ground into a powder using a grinder. The powder was then dissolved in normal saline (0.9%) to obtain a final concentration of 200 mg/mL.
5. Black seed gavage
In the treatment group, following induction of hyperthermia, animals received black seed at 300 mg/kg orally once daily for 35 days and were subsequently euthanized for histological and molecular analyses [13].
6. Sperm parameters
For sperm analysis, the mice were first anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and were then euthanized by cervical dislocation. The epididymal tails were isolated, and the semen was transferred into Ham’s F-10 medium (Sigma-Aldrich, Product No. N6635). The samples were then incubated at 37 °C for 20 minutes. Finally, 10 μL of semen was placed on a slide to evaluate sperm motility, and sperm count was determined using a counting chamber [14].
7. Histological studies
1) Tissue preparation
For tissue preparation, the testes (n=5) were isolated and immersed in Bouin’s solution for 48 hours. After fixation, the samples underwent dehydration and embedding using a tissue processor. The paraffin blocks were then sectioned at 5 and 10 μm thicknesses, and the sections were stained with hematoxylin and eosin for histological examination.
2) Testicular volume
Stereological evaluations were performed using established quantitative methods. Testicular volume was estimated according to the ‘Cavalieri principle’ using the following formula [15]:
where ΣP represents the total number of points intersecting the testicular sections, a/p denotes the area associated with each point, and t is the distance between consecutive sampled sections.
3) Quantity of germ cells and somatic cells
The total numbers of germ cells and somatic cells were determined using the ‘optical disector’ method according to the following formula [15]:
where ΣQ is the total number of counted cells, ΣP is the number of points intersecting the selected fields, a/f is the frame area, h is the disector height, BA is the microtome section thickness, and t is the actual measured section thickness.
8. Pro-inflammatory cytokines: TNF-α and IL-1β
To determine the levels of TNF-α and interleukin 1β (IL-1β) in testicular tissue, a commercial enzyme-linked immunosorbent assay (ELISA) kit (Milenia Biotech) was used according to the manufacturer’s instructions. Briefly, the assay was performed as a solid-phase ELISA on a microplate coated with monoclonal antibodies specific for TNF-α and IL-1β. After addition of a second horseradish peroxidase-labeled polyclonal antibody directed against a different epitope of TNF-α or IL-1β, a sandwich complex was formed between the two antibodies and the target cytokine. The chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was then added, and the optical density of the colored end product was measured at 450 nm using a microplate reader [16].
9. Gene expression of Bax, Bcl-2, and Caspase-3
Tissue samples were immediately placed in an RNA-stabilizing solution to preserve RNA integrity and were subsequently stored at −80 °C. Total RNA was extracted using an RNA isolation kit (RNX-Plus; Cinna Gen Co.) according to the manufacturer’s instructions. The RNA was then treated with DNase I (Roche) to remove genomic DNA. Complementary DNA was synthesized at 42 °C for 60 minutes using a commercial kit. The expression levels of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and caspase-3 were quantified by SYBR Green real-time polymerase chain reaction using exon-exon junction-specific primers designed with Primer3Plus software and validated with NCBI Primer-BLAST (Table 1). At the end of the experiment, threshold cycle (Ct) values, together with amplification and melting curves, were collected for analysis. Relative gene expression was calculated using the 2-ΔΔCt method.
10. Measurement of oxidative stress markers
Testicular tissue was processed using trypsin-ethylenediaminetetraacetic acid, washed with phosphate-buffered saline, and centrifuged at 1,200 rpm at 4 °C for 5 minutes. Intracellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescein diacetate (DCFDA; 20 μM) staining, followed by incubation at 37 °C for 45 minutes in the dark. Spectrofluorometric analysis was then performed using an excitation wavelength of 488 nm.
11. Measurement of GSH levels
Glutathione (GSH) levels were measured using a spectrofluorometric method. Testicular cell suspensions (0.5 mL) were treated with the fluorescent probes O-phthalaldehyde (OPA) and N-ethylmaleimide (NEM). After staining, the cells were centrifuged at 1,000 rpm for 1 minute, and the resulting pellet was resuspended in 2 mL of fresh culture medium. The cells were washed twice to remove residual fluorescent dye. Fluorescence intensity was then measured using a Shimadzu RF-5000U spectrophotometer with excitation and emission wavelengths set at 495 and 530 nm, respectively [17].
12. Statistical analysis
Statistical analysis was performed using SPSS ver. 27 (IBM Co.). Data normality was assessed with the Shapiro-Wilk test. Pairwise comparisons among groups were conducted utilizing Tukey’s and Dunn’s post hoc tests. Graphs were generated using Prism ver. 10 (GraphPad Software Inc.). All data are presented as mean±standard deviation, and a p-value <0.05 was considered statistically significant.
Results
1. Black seed increased the sperm count and sperm motility following scrotal hyperthermia
The results showed that sperm count and motility were markedly reduced following scrotal hyperthermia. However, administration of black seed significantly improved both sperm count and motility in the Hyper+BS group compared with the hyperthermia group (p≤0.0001 and p=0.0266, respectively) (Table 2, Figure 1).
2. Black seed improved the testicular volume following scrotal hyperthermia
The hyperthermia group showed a marked reduction in testicular volume, likely reflecting germ cell apoptosis and damage to the interstitial compartment of the seminiferous tubules. In contrast, black seed administration significantly improved testicular volume compared with the hyperthermia group (p=0.0166) (Table 2, Figure 2).
3. Black seed increased the number of Sertoli cells and germ cells, and did not change the number of Leydig cells following scrotal hyperthermia
Histological evaluation showed that heat stress significantly reduced the numbers of Sertoli cells and germ cells. However, after black seed administration, the numbers of these cells were significantly increased compared with those in the hyperthermia group. In contrast, no significant differences in Leydig cell number were observed among the groups (p=0.04, p=0.0288, p≤0.0001, p=0.0066, and p=0.7385, respectively) (Table 2, Figure 3).
5. Black seed decreased the expression of caspase-3 and increased the expression of Bcl-2 following scrotal hyperthermia
The findings indicated that Bax and caspase-3 expression levels were significantly increased in the hyperthermia group, whereas Bcl-2 expression was significantly decreased. Following black seed administration, caspase-3 expression was significantly reduced in the Hyper+BS group compared with the hyperthermia group. In contrast, Bax expression did not differ significantly between the Hyper+BS and hyperthermia groups. Furthermore, Bcl-2 expression was significantly increased in the Hyper+BS group relative to the hyperthermia group (p=0.0012, p=0.2291, and p=0.0038, respectively) (Table 3, Figure 5).
Discussion
The results of this study showed that black seed administration significantly increased the numbers of germ cells and Sertoli cells, testicular volume, sperm count, sperm motility, GSH levels, and Bcl-2 expression, while significantly decreasing pro-inflammatory cytokine levels, ROS levels, and caspase-3 expression in testicular tissue. However, no significant changes were observed in Leydig cell number or Bax expression following scrotal hyperthermia.
Scrotal hyperthermia has been associated with reproductive toxicity, including germ cell apoptosis, oxidative stress, inflammation, and reduced sperm quality [18]. Identifying herbal agents with protective effects and acceptable safety profiles is therefore of considerable interest [19]. In the present study, black seed (N. sativa) appeared to attenuate these abnormalities, likely through its antioxidant, anti-inflammatory, and anti-apoptotic properties.
The testes are highly sensitive to elevated temperatures, and both chronic and transient hyperthermia can activate apoptotic pathways in germ cells. Sertoli cells play a critical role in maintaining testicular architecture, and exposure to high temperatures can impair their function or lead to cell death [12,20]. Hyperthermia stimulates TNF-α receptor signaling in Sertoli and Leydig cells, activates the NF-κB pathway, and increases FASL expression, ultimately promoting germ cell apoptosis and reducing testicular volume [4,12]. In our study, black seed significantly reduced TNF-α and IL-1β levels. This reduction in inflammatory cytokines was accompanied by increases in Sertoli cell and germ cell numbers and partial restoration of testicular structure [21]. These findings suggest that the anti-inflammatory compounds present in black seed may help restore Sertoli cell function, improve structural and nutritional support, and create favorable conditions for germ cell survival and growth [11].
ROS disrupt cellular homeostasis in germ cells and play a central role in testicular inflammation. Because oxidative stress can stimulate the expression of inflammatory mediators, it may initiate an oxido-inflammatory cycle in testicular tissue [22]. Hyperthermia increases ROS production, which in turn upregulates TNF-α and IL-1β expression. Heat stress can also directly activate NF-κB, further increasing inflammatory mediator levels [23]. In the present study, black seed administration significantly reduced ROS levels, thereby attenuating oxidative stress and inflammation in testicular tissue. The bioactive compounds in black seed may reduce inflammation by lowering oxidative stress, decreasing the expression of inflammatory enzymes such as inducible nitric oxide synthase, and improving Sertoli cell function [11]. This antioxidant effect may also have contributed to the observed improvements in germ cell survival and spermatogenic function, which were supported by histological findings [23]. Consistent with our findings, Hadi et al [24]. also reported that N. sativa reduces inflammatory responses by modulating oxidative and inflammatory pathways.
We found that black seed was associated with a significant reduction in caspase-3 expression and a substantial increase in Bcl-2 expression, whereas Bax expression did not change significantly. These results suggest that black seed may help prevent germ cell death by modulating the oxidative-inflammatory cycle, thereby improving testicular structure. Consistent with our observations, previous studies have reported that N. sativa exhibits anti-apoptotic activity through modulation of oxidative and inflammatory pathways [25].
Thymoquinone, the principal active compound of N. sativa, appears to suppress oxidative stress and inflammation, increase Bcl-2 expression, and reduce apoptotic activity in testicular tissue. By improving the survival and number of testicular cells, it may help preserve tissue structure, maintain spermatogenesis, and enhance sperm function [26]. These findings are consistent with the study by Ardabili et al. [27], which reported that N. sativa improves testicular injury through modulation of oxidative, inflammatory, and apoptotic pathways.
This study had several limitations, including the use of an animal model rather than human subjects. Although mice share important physiological similarities with humans in reproductive biology, further validation is needed. In addition, protein expression was not examined, which limits mechanistic interpretation. Nevertheless, this study provides useful insight into the potential of black seed as a natural therapeutic candidate for male infertility. Scrotal hyperthermia disrupts spermatogenesis by inducing oxidative stress, inflammation, and apoptosis. In this model, black seed administration reduced ROS, caspase-3, and TNF-α levels while preserving the structure of the spermatogenic epithelium, thereby improving testicular structure and function. These findings suggest that black seed may have potential as a natural agent for preserving fertility following environmental stress, although further research is required. Future studies should explore different doses and treatment durations of black seed and, where feasible, extend these findings to clinical studies in humans to confirm efficacy and safety in male reproductive health.
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