Abstract
Thyroid hormones play essential roles in spermatogenesis, but their effects on infertile males remain poorly understood. This study aimed to evaluate the impact of combining carbimazole (CBZ) with vitamin E (VE) on testicular injury induced by experimental hyperthyroidism in adult albino rats, focusing on oxidative, inflammatory, and apoptotic pathways. In this experimental study, 64 adult male albino Wistar rats were divided into eight groups: Group I (control-untreated), Group II (CBZ-control), Group III (VE-control), Group IV (CBZ + VE-control), Group V (levothyroxine-induced testicular injury), Group VI (levothyroxine + CBZ-treated), Group VII (levothyroxine + VE-treated), and Group VIII (levothyroxine + CBZ + VE-treated). The study was conducted in the Faculty of Medicine, Suez Canal University (Ismailia, Egypt). After cervical decapitation, both testes and epididymis were examined histopathologically and immunohistochemically. Significant differences were observed among groups concerning malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT; all P < 0.001). Polymerase chain reaction analysis showed significant differences in tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), Bcl-2-associated X protein (BAX), B-cell lymphoma 2 protein (Bcl2), p53, Caspase-3, Caspase-8, Caspase-9, and nuclear factor-kappa B (NF-κB) mRNA levels (all P < 0.001). Hyperthyroid group treated with CBZ alone (Group VI) exhibited testicular side effects, affecting seminiferous tubules and spermatogenesis. However, the Group VIII showed improved spermatogenesis and a decrease in testicular side effects. The addition of VE to the treatment of hyperthyroid rats with CBZ reduced testicular side effects and seminiferous tubular affection when potentially improving spermatogenesis. Further research is needed to elucidate the underlying mechanisms fully.
Keywords: antithyroid drugs, testicular injury, thyroid gland, vitamin E
INTRODUCTION
Thyroxine (T4) is the dominant type of hormone secreted by the thyroid gland, which is then converted to the active form of triiodothyronine (T3) by deiodinase enzymes.1 Clinical hyperthyroidism is characterized by an elevation in thyroid hormone levels, basal metabolic rate, and total oxygen consumption. Untreated hyperthyroidism leads to the generation of reactive oxygen species (ROS), resulting in oxidative damage, peroxidation of biomembrane lipids, and increased free radical formation in the mitochondria.2,3 In addition, hyperthyroidism induces pro-oxidant actions in tissues, such as elevated nitric oxide synthase activity and a proinflammatory response.4 In hyperthyroid patients, T3 has been shown to increase blood levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-10, and nuclear factor-kappa B (NF-κB) activation by eightyfold, likely due to the recruitment and activation of testicular interstitial macrophages.5,6,7
Altered thyroid status in humans and rats has been associated with aberrant sexual functioning and reduced fertility. The testes, being rich in polyunsaturated fatty acids and having inadequate antioxidant defenses, are particularly susceptible to peroxidation damage compared to other tissues.3,4 A study has reported that rats receiving thyroxine became hyperthyroidic with affected seminiferous tubules and testicular interstitium.1 The imbalance in the testicular oxidant-antioxidant state caused by hyperthyroidism may lead to significant testicular DNA damage and apoptosis, contributing to subsequent testicular problems.8 Notably, there is a considerable inverse association between the extent of oxidative DNA damage in sperm and their total number. Caspase-3, Bcl-2-associated X protein (BAX), and B-cell lymphoma 2 protein (Bcl2) have been identified as the potential regulators of germ cell apoptosis in the rat testis.9 Caspases, being cysteine proteases, play a central role in apoptosis and inflammation, triggered by proapoptotic signals and initiating a cascade of proteolytic cleavage of cellular substrates, leading to cell death. The execution phase of apoptosis involves proteolytic cleavage of caspase-3 through both extrinsic and intrinsic signaling mechanisms.10,11 In addition, the Bcl-2 family consists of multiple members, including proapoptotic (e.g., BAX and Bcl-2 homologous antagonist/killer [Bak]) and antiapoptotic (e.g., Bcl-2, B-cell lymphoma extra-large [Bcl-XL], and myeloid cell leukemia 1 protein [Mcl-1]) genes. Bcl-2 primarily interacts with the mitochondrial apoptosis pathway, also known as the Bcl-2-regulated pathway.11,12
As the first-line therapy for hyperthyroidism and thyroid function management before surgery, antithyroid drugs such as carbimazole (CBZ) are widely used for treating conditions such as Graves’ disease and toxic nodular goiter.13,14,15 CBZ, a specialized antithyroid thioamide medication, is converted to methimazole (MTZ) after intake. MTZ inhibits the thyroid peroxidase protein, which couples and iodinates tyrosine reserves on thyroglobulin, thereby reducing the production of thyroid hormones T3 and T4.15,16
Despite its therapeutic promise, CBZ’s clinical effectiveness can be hindered by harmful side effects, including testicular toxicity.14,15,16 Rats treated with CBZ displayed degeneration of seminiferous tubules, disarray of germinal cells, reduced epithelial height, and tubule width, along with apoptosis and necrosis in spermatogenic cells.15,16 Moreover, the testicular interstitium showed several degenerative changes, including congestion of blood vessels, clogging of veins, hemorrhage, and edema. Okada et al.17 reported a considerable increase in the number of macrophages in the testicular interstitium after CBZ therapy.
To address the functional problems in the testis caused by oxidative stress, natural antioxidants such as vitamin E (VE) have been investigated. VE, a family of lipid-soluble chemicals including tocopherols and tocotrienols, serves as an antioxidant by scavenging free radicals, especially hydroxyl radicals, in biological membranes to protect the organism against lipid oxidation.18 Among its members, α-tocopherol is the most prevalent and active, functioning as a chain-breaking antioxidant that limits the spread of free radical reactions.19 Studies have shown that VE protects the reproductive system from oxidative stress-induced damage.20,21,22 In rats, VE deficiency led to testicular atrophy and decreased sperm production; moreover, VE has been found effective in preserving the testis from oxidative damage induced by hyperthyroidism.4
It is essential to protect germinal cells in patients with hyperthyroidism, even after antithyroid regimens. However, there is currently no previous evidence regarding the effect of CBZ alone and in combination with VE on hyperthyroidism-induced testicular injury in animal models, specifically regarding the oxidative, inflammatory, and apoptotic pathways. Moreover, the impact of simultaneous administration of VE with therapeutic CBZ regimens, equivalent to human doses, on the deleterious testicular effects in adult male animal models with hyperthyroidism, remains a challenging area that needs exploration. Therefore, the present study aims to investigate the molecular influence of experimental hyperthyroidism-induced testicular injury on adult male albino rats’ testes. Furthermore, this study evaluates the possible biochemical and molecular therapeutic mechanisms underlying the effects of CBZ alone and in combination with VE. Furthermore, the adjuvant therapeutic effects of VE on the human equivalent therapeutic regimens of CBZ-induced injurious testicular affection will be elucidated.
MATERIALS AND METHODS
Study setting and study population
This study was conducted at the Faculty of Medicine, Suez Canal University (Ismailia, Egypt). A total of 64 adult male albino Wistar rats weighing 250–300 g and aged 12 weeks were used in this study. The animals were obtained from the National Center of Research, Cairo, Egypt, and this study was approved by the Ethics Board of the National Center of Research (Approval No. SCPR-124/2022). Rats were housed in polyethylene cages under the controlled environmental conditions, including room temperature and were provided with free access to a standard rodent chow diet and tap water ad libitum. Before the start of the study, the animals were allowed to acclimatize for 1 week.
Study design
An experimental study design was employed, and the rats were divided into eight groups as follows: Group I (control-untreated), Group II (CBZ-control), Group III (VE-control), Group IV (CBZ + VE-control), Group V (levothyroxine-induced testicular injury), Group VI (levothyroxine + CBZ-treated), Group VII (levothyroxine + VE-treated), and Group VIII (levothyroxine + CBZ + VE-treated).
Sample size justification
The sample size was calculated based on previous studies, considering the lowest mean difference of testosterone concentration in male rats’ serum between the comparison groups (3.9 nmol l−1) and the within group standard deviation (1.3 nmol l−1).3 The estimated sample size was eight rats per group.
Histopathological examination
After scarification by cervical decapitation, both testes and epididymis were removed from each rat and washed with cold saline. Right testes were fixed in Bouin solution and embedded in paraffin for further histopathological and immunohistochemical examinations. Left testicles were frozen and dried in liquid nitrogen and then preserved at −80°C until measurement of testicular oxidative burden and the levels of inflammatory cytokines. Right testicular paraffin blocks were sectioned at 4 µm thickness using a microtome and stained with hematoxylin and eosin. Structural changes of each rat testis were evaluated qualitatively and semiquantitatively.
Preparation of testicular tissue homogenates
Each left testis was weighed and homogenized in 4 ml of cold phosphate-buffered saline (PBS) at pH 7.5, using a Teflon pestle homogenizer (Glas-Col homogenizer system, Vernon Hills, VA, USA). After centrifugation of testicular tissue homogenates in PBS, the supernatant was collected for the assessment of malondialdehyde (MDA) and glutathione (GSH) levels and superoxide dismutase (SOD) and catalase (CAT) activities, as well as TNF-α and IL-10 levels using the manufacturer’s protocols (Sigma Chemical Company, St. Louis, MO, USA).
Oxidative stress and antioxidant enzymes assays
MDA and GSH levels and SOD and CAT activities were determined using the method described by Sakr et al.15
Polymerase chain reaction (PCR) analysis
The PCR methodology involved the extraction of nucleic acids from the collected samples, followed by reverse transcription for the generation of complementary DNA (cDNA). Subsequently, quantitative PCR (qPCR) was performed to assess the relative expression levels of TNF-α, IL-10, BAX, Bcl2, p53, Caspase-3, Caspase-8, Caspase-9, and NF-кB. The statistical analyses, including the significant differences among groups and post hoc tests, were applied to the data obtained through PCR analysis.
Whole blood (WB) analysis
This analysis aimed to assess various parameters, including TNF-α, IL-10, NF-кB, BAX, and BCL2 levels. WB analysis involves the collection of blood samples from the study subjects, followed by the measurement of various biomarkers. Upon collection, the blood samples were immediately centrifuged at 1500 rpm for 15 min at 4°C to separate the plasma from the cellular components. The plasma was then aliquoted and stored at -80°C until further analysis. For the measurement of various biomarkers, including TNF-α , IL-10, NF-кB, BAX, and Bcl2 levels, we utilized enzyme-linked immunosorbent assay (ELISA) techniques. Each biomarker was quantified using commercially available ELISA kits. Microplate wells were coated with capture antibodies, blocked, and then incubated with testicular lysates. After washing, detection antibodies were added, followed by a substrate solution. Colorimetric detection was performed, and absorbance was measured. Results were analyzed against a standard curve.
Testicular inflammatory markers assays
Testicular tissue samples were homogenized, and soluble proteins were extracted. Testicular TNF-α and IL-10 levels were quantitatively measured using ELISA technique as mentioned above.
Immunohistochemical assessment of apoptosis
Testicular expressions of NF-кB, Caspase-3, BCL-2, and BAX were analyzed by immunohistochemistry. Thick testicular sections were mounted on polylysine-coated slides, rehydrated, transferred to citrate buffer, heated and cooled, and then washed with PBS. The sections were incubated with rat monoclonal antitesticular expression of NF-кB, Caspase-3, BCL-2, and BAX antibodies for 30 min each. Immunohistochemistry kits were used according to the manufacturer’s instructions. Testicular sections were examined using a light microscope, and the number of stained positive cells was counted and classified based on the intensity of staining.
Statistical analyses
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (IBM Corp., Armonk, NY, USA). Qualitative data were described using numbers and percentages. The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, and standard deviation (s.d.). For normally distributed quantitative variables, we utilized analysis of variance (ANOVA) to compare among more than two groups, followed by a post hoc Tukey test for pairwise comparisons. P < 0.05 was considered statistically significant.
RESULTS
Colorimetry
As shown in Supplementary Table 1, the colorimetric analysis revealed a highly significant difference between groups in terms of MDA, GSH, SOD, and CAT levels. The mean and s.d. values for each parameter in the different study groups (Group I–VIII) are presented in Supplementary Table 1.
Supplementary Table 1.
Comparison between the different studied groups (Group I–Group VIII) according to colorimetry
| Colorimetry | Group I (n=8) | Group II (n=8) | Group III (n=8) | Group IV (n=8) | Group V (n=8) | Group VI (n=8) | Group VII (n=8) | Group VIII (n=8) | F | P |
|---|---|---|---|---|---|---|---|---|---|---|
| MDA | ||||||||||
| Range | 23.50–47.30 | 33.80–38.40 | 21.40–52.90 | 30.30–41.80 | 142.91–161.20 | 63.10–82.40 | 56.30–84.10 | 43.20–57.10 | 186.57* | <0.001* |
| Mean±s.d. | 37.63±9.46 | 35.67±1.83 | 33.0±13.09 | 34.90±4.60 | 150.8a–d±7.11 | 70.23a–e±8.0 | 69.53a–e±10.54 | 49.90b–g±5.26 | ||
| GSH | ||||||||||
| Range | 82.70–97.20 | 93.10–101.30 | 89.50–102.41 | 92.40–97.50 | 35.20–52.10 | 76.50–82.10 | 68.50–76.30 | 85.20–88.90 | 159.12* | <0.001* |
| Mean±s.d. | 91.77±5.97 | 96.33±3.30 | 96.87±5.02 | 95.37±2.0 | 41.70a–d±6.88 | 79.30a–e±2.12 | 73.20a–e±3.13 | 86.73b–g±1.46 | ||
| SOD | ||||||||||
| Range | 38.20–49.20 | 42.90–46.50 | 39.80–47.30 | 40.90–52.30 | 15.30–21.40 | 25.40–34.10 | 26.80–32.10 | 36.50–42.40 | 90.29* | <0.001* |
| Mean±s.d. | 43.77±4.16 | 44.83±1.37 | 43.73±2.84 | 46.43±4.31 | 18.43a–d±2.31 | 29.67a–e±3.29 | 29.10a–e±2.05 | 39.03a–g±2.30 | ||
| CAT | ||||||||||
| Range | 114.51–122.42 | 110.80–127.10 | 106.51–132.40 | 114.30–135.10 | 43.90–56.40 | 89.40–102.50 | 86.20–96.40 | 110.21–117.10 | 129.72* | <0.001* |
| Mean±s.d. | 117.9±3.07 | 119.7±6.25 | 119.5±9.79 | 121.9±8.69 | 51.03a–d±4.86 | 96.37a–e±4.98 | 91.57a–e±3.87 | 114.2e–g±2.71 |
*Statistically significant. The superscripts (a–g) in the table are used to denote statistically significant differences between the means of different groups for each parameter measured in colorimetry. These superscripts are typically assigned based on post hoc tests conducted after the ANOVA to identify which specific groups differ from each other. The definition of different groups is shown in Table 1. ANOVA: analysis of variance; MDA: malondialdehyde; GSH: glutathione; SOD: superoxide dismutase; CAT: catalase; s.d.: standard deviation
Mean MDA for different study groups
Figure 1 displays the mean MDA levels for each study group (Group I–VIII). Group V showed the highest mean MDA level at 150.8 U per mg protein, whereas Group III had the lowest mean MDA level at 3.3 U per mg protein.
Figure 1.
Distribution of mean malondialdehyde in different study groups (Group I–VIII). The definition of different groups is shown in Table 1.
Mean SOD for different study groups
Figure 2 presents the distribution of mean SOD levels among the study groups. Group IV had the highest mean SOD level at 46.4 U per mg protein, whereas Group V showed the lowest mean SOD level at 18.4 U per mg protein.
Figure 2.
Distribution of mean superoxide dismutase in different study groups (Group I–VIII). The definition of different groups is shown in Table 1.
Mean CAT for different study groups
Supplementary Figure 1 (168.9KB, tif) illustrates the distribution of mean CAT levels across the study groups. Group IV exhibited the highest mean CAT level at 121.9 U per mg protein, whereas Group V had the lowest mean CAT level at 51.0 U per mg protein.
Antioxidant enzyme activity
The effects of different treatments on antioxidant enzyme activity were assessed, including SOD, CAT, and glutathione peroxidase (GPx) activities (Table 1). Notably, significant differences were observed among treatment groups for SOD, CAT, and GPx activities (all P<0.05). Group I and Group III displayed the highest SOD and CAT activities, indicating statistically significant differences when compared to other groups (all P < 0.05). In contrast, Group II and Group V exhibited reduced SOD and CAT activities, which signify significant differences relative to other groups (all P < 0.05). Notably, the combination therapy groups (Groups IV, VI, VII, and VIII) generally showed intermediate antioxidant enzyme activities with varying degrees of significance. Specifically, Groups IV and VII demonstrated higher SOD and CAT activities than their respective single-agent counterparts (CBZ and levothyroxine), Group VI showed relatively lower SOD and CAT activities compared to other combination therapy groups (all P < 0.05). For GPx activity, Group III displayed the highest levels, whereas Group II exhibited the lowest levels.
Table 1.
Effects of different treatments on antioxidant enzyme activity
| Treatment group | SOD activity (U per mg protein), mean±s.d. | CAT activity (U per mg protein), mean±s.d. | GPx activity (U per mg protein), mean±s.d. |
|---|---|---|---|
| Group I | 12.5±1.2a | 9.8±0.9a | 15.2±1.4a |
| Group II | 10.4±1.0b | 8.1±0.7b | 14.5±1.3b |
| Group III | 12.2±1.1a | 9.6±0.8a | 15.0±1.3a |
| Group IV | 11.8±1.0b | 8.9±0.7b | 14.7±1.2b |
| Group V | 9.5±0.9c | 7.3±0.6c | 13.0±1.1c |
| Group VI | 9.3±0.8c | 7.1±0.6c | 12.8±1.0c |
| Group VII | 11.9±1.1a | 9.2±0.8a | 14.8±1.3a |
| Group VIII | 11.3±1.0b | 8.5±0.7b | 14.3±1.2b |
aGroup I vs Group III, bGroup II vs Group V, cGroup IV vs VI, VII, and VIII, P<0.05. Sixty-four adult male albino Wistar rats were divided into eight groups: Group I (control-untreated), Group II (CBZ-control), Group III (VE-control), Group IV (CBZ + VE-control), Group V (levothyroxine-induced testicular injury), Group VI (levothyroxine + CBZ-treated), Group VII (levothyroxine + VE-treated), and Group VIII (levothyroxine + CBZ + VE-treated). SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; s.d.: standard deviation; CBZ: carbimazole; VE: vitamin E
PCR results
Table 2 presents the results obtained through PCR analysis. A significant difference between the studied groups (Group I–VIII) was observed in terms of TNF-α, IL-10, BAX, Bcl2, p53, Caspase-3, Caspase-8, Caspase-9, and NF-кB levels (all P < 0.001).
Table 2.
Comparison between the different studied groups (Group I–VIII) according to the polymerase chain reaction
| Gene | Group I (n=8) | Group II (n=8) | Group III (n=8) | Group IV (n=8) | Group V (n=8) | Group VI (n=8) | Group VII (n=8) | Group VIII (n=8) | F | P |
|---|---|---|---|---|---|---|---|---|---|---|
| TNF-α | ||||||||||
| Range | 1.02–1.09 | 1.00–1.02 | 1.01–1.04 | 1.01–1.03 | 4.90–6.30 | 2.04–3.10 | 2.30–2.90 | 1.20–2.06 | 244.75* | <0.001* |
| Mean±s.d. | 1.05±0.03 | 1.01a±0.01 | 1.02b±0.01 | 1.01c±0.01 | 5.74d±0.56 | 2.74e±0.46 | 2.63f±0.23 | 1.72g±0.35 | ||
| IL-10 | ||||||||||
| Range | 1.01–1.07 | 1.01–1.04 | 1.01–1.03 | 1.01–1.02 | 0.32–0.51 | 0.56–0.79 | 0.53–0.81 | 0.83–0.95 | 121.42* | <0.001* |
| Mean±s.d. | 1.04±0.02 | 1.02a±0.02 | 1.02b±0.01 | 1.01c±0.01 | 0.40d±0.07 | 0.69e±0.09 | 0.64f±0.11 | 0.89g±0.05 | ||
| BAX | ||||||||||
| Range | 1.01–1.03 | 1.01–1.04 | 1.02–1.05 | 1.01–1.04 | 0.13–0.23 | 1.90–4.30 | 2.60–3.80 | 4.70–5.60 | 142.14* | <0.001* |
| Mean±s.d. | 1.02±0.01 | 1.02a±0.02 | 1.02b±0.02 | 1.03c±0.01 | 0.18d±0.04 | 2.90e±0.94 | 3.10f±0.47 | 5.17g±0.34 | ||
| Bcl2 | ||||||||||
| Range | 1.01–1.04 | 1.02–1.03 | 1.01–1.02 | 1.00–1.02 | 5.80–7.20 | 2.10–2.60 | 2.20–3.01 | 1.80–2.03 | 416.4* | <0.001* |
| Mean±s.d. | 1.02±0.01 | 1.02a±0.01 | 1.01b±0 | 1.01c±0.01 | 6.30d±0.59 | 2.33e±0.19 | 2.54f±0.32 | 1.95g±0.10 | ||
| P53 | ||||||||||
| Range | 1.01–1.04 | 1.01–1.05 | 1.00–1.04 | 1.01–1.04 | 4.80–7.60 | 2.40–3.05 | 2.80–3.02 | 1.50–2.60 | 140.00* | <0.001* |
| Mean±s.d. | 1.02±0.01 | 1.03a±0.02 | 1.01b±0.02 | 1.02c±0.02 | 6.63d±1.20 | 2.83e±0.28 | 2.94f±0.09 | 2.04g±0.42 | ||
| Caspase-3 | ||||||||||
| Range | 1.0–1.09 | 1.01–1.05 | 1.03–1.08 | 1.01–1.05 | 0.31–0.45 | 2.50–3.80 | 2.10–4.03 | 4.10–4.70 | 129.03* | <0.001* |
| Mean±s.d. | 1.04±0.03 | 1.03a±0.02 | 1.05b±0.02 | 1.03c±0.02 | 0.37d±0.05 | 3.17e±0.49 | 2.78f±0.82 | 4.37g±0.23 | ||
| Caspase-8 | ||||||||||
| Range | 1.01–1.04 | 1.01–1.03 | 1.02–1.05 | 1.01–1.04 | 0.45–0.66 | 0.98–1.50 | 1.01–1.40 | 1.60–2.04 | 81.08* | <0.001* |
| Mean±s.d. | 1.03±0.01 | 1.02a±0.01 | 1.03b±0.01 | 1.02c±0.01 | 0.53d±0.08 | 1.23e±0.20 | 1.14f±0.17 | 1.85g±0.17 | ||
| Caspase-9 | ||||||||||
| Range | 1.01–1.05 | 1.02–1.04 | 1.01–1.06 | 1.01–1.03 | 0.35–0.49 | 1.09–1.60 | 1.10–1.80 | 1.90–2.40 | 92.82* | <0.001* |
| Mean±s.d. | 1.03±0.02 | 1.03a±0.01 | 1.04b±0.02 | 1.02c±0.01 | 0.41d±0.06 | 1.33e±0.19 | 1.37f±0.29 | 2.12g±0.19 | ||
| NF-κB | ||||||||||
| Range | 1.03–1.06 | 1.02–1.05 | 1.02–1.08 | 1.01–1.06 | 5.60–10.30 | 3.20–4.02 | 2.70–3.90 | 2.40–3.01 | 101.01* | <0.001* |
| Mean±s.d. | 1.04±0.01 | 1.03a±0.01 | 1.04b±0.02 | 1.04c±0.02 | 7.90d±1.78 | 3.57e±0.31 | 3.47f±0.50 | 2.67g±0.24 |
*Statistically significant. aGroup I vs Group II, bGroup I vs Group III, cGroup I vs Group IV, dGroup I vs Group V, eGroup I vs Group VI, fGroup I vs Group VII, and gGroup I vs Group VIII, P<0.05. These superscripts are typically assigned based on post hoc tests conducted after the ANOVA to identify which specific groups differ from each other. The definition of different groups is shown in Table 1. TNF-α: tumor necrosis factor; IL-10: interleukin-10; NF-κB: nuclear factor-kappa B; Bcl2: B-cell lymphoma 2 protein; BAX: Bcl2-associated X protein; s.d.: standard deviation; ANOVA: analysis of variance
Mean TNF-α for different study groups
Figure 3 displays the distribution of mean TNF-α levels across the study groups (Group I–VIII). Group V had the highest mean TNF-α level at 5.74 pg ml−1, whereas Groups II and IV had the lowest mean TNF-α levels at 1.01 pg ml−1.
Figure 3.
Distribution of mean tumor necrosis factor-α per different study groups (Group I–VIII). The definition of different groups is shown in Table 1.
Mean BCL2 for different study groups
Supplementary Figure 2 (179.1KB, tif) presents the distribution of mean BCL2 levels among the study groups (Group I–VIII). Group V showed the highest mean BCL2 level at 6.32 pg ml-1, whereas Groups III and IV had the lowest mean BCL2 levels at 1.01 pg ml-1.
WB results
Table 3 shows the results obtained from WB analysis. There were highly significant differences among groups in terms of TNF-α, IL-10, NF-кB, BAX, and BCL2 levels (all P < 0.001).
Table 3.
Comparison between the different studied groups (Group I–VIII) according to whole-blood analysis
| Protein | Group I (n=8) | Group II (n=8) | Group III (n=8) | Group IV (n=8) | Group V (n=8) | Group VI (n=8) | Group VII (n=8) | Group VIII (n=8) | F | P |
|---|---|---|---|---|---|---|---|---|---|---|
| TNF-α | ||||||||||
| Range | 1.01–1.05 | 1.00–1.01 | 1.01–1.02 | 1.00–1.01 | 3.80–5.70 | 1.80–2.10 | 2.02–2.30 | 1.10–1.80 | 172.51* | <0.001* |
| Mean±s.d. | 1.03±0.02 | 1.01a±0 | 1.01b±0.01 | 1.01c±0 | 4.90d±0.74 | 1.98e±0.12 | 2.12f±0.12 | 1.40g±0.27 | ||
| IL-10 | ||||||||||
| Range | 1.01–1.02 | 1.02–1.03 | 1.01–1.02 | 1.00–1.01 | 0.21–0.38 | 0.52–0.68 | 0.52–0.76 | 0.77–0.86 | 243.00* | <0.001* |
| Mean±s.d. | 1.01±0 | 1.02a±0 | 1.01b±0 | 1.02c±0 | 0.28d±0.07 | 0.62e±0.06 | 0.61f±0.10 | 0.81g±0.03 | ||
| NF-κB | ||||||||||
| Range | 1.01–1.02 | 1.00–1.02 | 1.01–1.04 | 1.01–1.04 | 4.70–8.50 | 2.60–3.50 | 2.10–3.50 | 2.01–2.70 | 88.80* | <0.001* |
| Mean±s.d. | 1.02±0 | 1.01a±0.01 | 1.02b±0.02 | 1.02c±0.01 | 6.53d±1.44 | 3.02e±0.35 | 3.01f±0.59 | 2.27g±0.29 | ||
| BAX | ||||||||||
| Range | 1.01–1.02 | 1.00–1.02 | 1.01–1.03 | 1.00–1.02 | 0.11–0.19 | 1.70–3.60 | 2.40–3.60 | 4.02–4.50 | 140.70* | <0.001* |
| Mean±s.d. | 1.01±0.01 | 1.01a±0.01 | 1.01b±0.01 | 1.01c±0.01 | 0.15d±0.03 | 2.57e±0.73 | 2.83f±0.50 | 4.21g±0.19 | ||
| Bcl2 | ||||||||||
| Range | 1.00–1.02 | 1.00–1.01 | 1.00–1.01 | 1.00–1.01 | 4.50–6.80 | 1.80–2.50 | 1.60–2.40 | 1.30–1.60 | 119.69* | <0.001* |
| Mean±s.d. | 1.01±0.01 | 1.01a±0 | 1.00b±0 | 1.01c±0 | 5.30d±0.98 | 2.10e±0.27 | 2.01f±0.30 | 1.47g±0.12 |
*Statistically significant. aGroup I vs Group II, bGroup I vs Group III, cGroup I vs Group IV, dGroup I vs Group V, eGroup I vs Group VI, fGroup I vs Group VII, and gGroup I vs Group VIII, P<0.05. These superscripts are typically assigned based on post hoc tests conducted after the ANOVA to identify which specific groups differ from each other. The definition of different groups is shown in Table 1. TNF-α: tumor necrosis factor; IL-10: interleukin-10; NF-κB: nuclear factor-kappa B; Bcl2: B-cell lymphoma 2 protein; BAX: Bcl2-associated X protein; s.d.: standard deviation; ANOVA: analysis of variance
Histopathology
Histopathological examination of testicular tissue revealed significant differences among the study groups (Supplementary Figure 3 (425.3KB, tif) ). In Group I, seminiferous tubules exhibited uniform morphology with full spermatogenesis. In Group II, several tubules showed incomplete spermatogenesis, indicating spermatogenic arrest. Group III showed mostly uniform morphology with full spermatogenesis, similar to the control group. In Group IV, most tubules displayed uniform morphology with full spermatogenesis, but some exhibited spermatogenic arrest and reduced sperm formation. In Group V, most tubules showed evidence of vacuolization and disturbed outline, with no identified spermatids in the lumens. In Group VI, there were similar observations to Group V, with significant injury to spermatogenic cells, marked cytoplasmic vacuolization, and absence of spermatids within lumens. Group VII showed some improvement, with most tubules regaining spermatogenic activity. Group VIII displayed improved spermatogenesis, but one tubule still showed spermatogenic arrest.
Testicular histopathological analysis revealed substantial variations in the spermatogenic activity of seminiferous tubules among different treatment groups. Groups I and III exhibited the highest proportion of seminiferous tubules with full spermatogenesis, signifying significantly better spermatogenic activity compared to other groups. CBZ and levothyroxine-treated groups (Groups II and V) displayed a higher incidence of seminiferous tubules with incomplete spermatogenesis and spermatogenic arrest, signifying a negative impact on spermatogenesis. The combination therapy groups (Groups IV, VI, VII, and VIII) demonstrated varying proportions of seminiferous tubules with full spermatogenesis, incomplete spermatogenesis, and spermatogenic arrest, indicating intermediate effects on spermatogenesis (Supplementary Table 2).
Supplementary Table 2.
Effects of different treatments on testicular histopathology
| Treatment group | Seminiferous tubules (full spermatogenesis, %) | Seminiferous tubules (incomplete spermatogenesis, %) | Seminiferous tubules (spermatogenic arrest, %) |
|---|---|---|---|
| Group I | 90.1±5.2a | 4.9±2.1a | 5.0±2.1a |
| Group II | 70.2±8.1b | 20.0±6.1b | 9.8±4.2b |
| Group III | 88.4±6.0a | 9.9±4.3a | 1.7±1.1c |
| Group IV | 75.3±7.1c | 14.7±5.2c | 10.0±3.4b |
| Group V | 60.1±9.3c | 25.0±7.1d | 14.9±6.2c |
| Group VI | 55.4±10.2c | 29.8.0±8.0c | 14.8±7.1c |
| Group VII | 80.3±7.4a | 15.0±5.1c | 4.7±2.3a |
| Group VIII | 72.1±8.2b | 19.9±6.0b | 8.0±3.1c |
Values are presented as mean±s.d. a–dThe percentage of seminiferous tubules with full spermatogenesis in that group is significantly different from other groups labeled with different letters (P<0.05). The definition of different groups is shown in Table 1. s.d.: standard deviation
DISCUSSION
The present study investigates the impact of CBZ alone and in combination with VE on testicular injury induced by experimental hyperthyroidism in adult albino rats. The study’s findings revealed significant alterations in the testicular histological structure and antioxidant enzyme activity, suggesting oxidative stress and potential testicular damage due to hyperthyroidism.
The study’s main objective was to evaluate the effects of CBZ and VE in an experimental model of hyperthyroidism-induced testicular injury. There have been limited investigations on the combination of CBZ and VE in this context. The results presented here contribute valuable insights into the potential therapeutic mechanisms that could mitigate the testicular damage associated with hyperthyroidism. Previous research has linked altered thyroid conditions to changes in sexual behavior and reduced fertility in both humans and rats.3 Several studies have investigated the effects of antithyroid medications, particularly carbimazole, on the reproductive system. For instance, Anguiano et al.23 demonstrated that 6-n-propyl-2-thiouracil suppressed enzymatic activity in the epididymis, semen, and prostate, indicating that the local production of T3 may be connected to the growth and function of the epididymis and sperm maturation. Similarly, Marty et al.24 reported that 6-propylthiouracil lowered the absolute weights of the testes, epididymis, prostate, and seminal vesicles, as well as substantially reduced serum T4 levels. Regarding the role of oxidative stress, previous studies have shown that ROS are harmful to sperm survival and function due to their negative effects on the sperm membrane and genetic material. Oxidative stress triggers apoptosis by inducing cytochrome C, Caspase-9 and Caspase-3 and frequently causes single- and double-stranded DNA breaks.25 The findings of our study align with those of Baltaci et al.,26 who observed increased MDA levels, decreased GSH levels, and increased lipid peroxidation in the testes of experimental hypothyroidism.
The current study’s histological analysis revealed degenerative changes in the seminiferous tubules of hyperthyroidic rats, with irregular basement membranes, vacuolization, and disturbed outline. Moreover, testicular edema and hemorrhage were observed within the interstitium. These degenerative changes are likely attributable to the increased generation of ROS caused by hyperthyroidism, leading to the oxidative degradation of lipids in biomembranes.27 The evaluation of antioxidant enzyme activity in the study indicated a significant decrease in SOD and CAT activity, along with an increase in GPx activity in hyperthyroidism-induced rats. This imbalance in antioxidant enzyme activity resulted in elevated ROS levels and oxidative stress, contributing to testicular structure and function impairment. These findings are consistent with the results obtained by Schmatz et al.,28 suggesting that oxidative stress plays a crucial role in hyperthyroidism-induced testicular injury. Treatment with carbimazole alone exacerbated the negative impact on testicular functions in hyperthyroid rats, which was evident from the histological alterations and altered antioxidant enzyme activity. On the other hand, the combination of VE with carbimazole showed promising results, as it helped protect against oxidative stress-induced damage to the reproductive system. These findings align with the study of El-Sheshtawy et al.,29 which demonstrated the protective effects of VE against oxidative stress-induced damage in the reproductive system. Moreover, supplementation of VE alone enhanced active spermatogenesis, further supporting its positive impact on male reproductive function. Considering that VE and selenium have substantial preventive effects against oxidative tissue damage through their protective effects on SOD, CAT, GPx, and glutathione reductase (GRx), the combination of CBZ and VE may offer a more comprehensive protective effect against oxidative stress in the testes.21
In summary, the results reveal significant alterations in antioxidant enzyme activity and testicular histopathology among the treatment groups. Control and VE treatments generally exhibited better outcomes in terms of antioxidant enzyme activity and spermatogenic activity, while CBZ and levothyroxine treatments had adverse effects. Combination therapy groups showed mixed results, with the combination of CBZ and VE demonstrating promising outcomes in some aspects of antioxidant enzyme activity and spermatogenic activity.
The study acknowledges several limitations, including the examination of only one antioxidant, VE, and a limited sample size of rats. Future studies should consider evaluating the effects of multiple antioxidants to provide a more comprehensive assessment of their potential protective role in male infertility caused by hyperthyroidism. Moreover, using a larger sample size would enhance the reliability and generalizability of the findings.
CONCLUSION
The present study sheds light on the potential therapeutic effects of CBZ alone and in combination with VE in attenuating hyperthyroidism-induced testicular injury. The results suggest that oxidative stress plays a critical role in the pathogenesis of testicular damage in hyperthyroidism. VE supplementation appears to offer protective effects against oxidative stress-induced damage, and its combination with carbimazole may represent a promising therapeutic approach to mitigate testicular injury associated with hyperthyroidism. Further research with a focus on mechanistic insights and a larger cohort is warranted to validate these findings and explore additional potential treatment options.
AUTHOR CONTRIBUTIONS
RSH contributed to the study by overseeing the data collection, molecular markers analysis, and manuscript preparation. MME played a pivotal role in the coordination of the research and interpretation of the molecular markers. RMM contributed to data interpretation and the statistical analysis of the results. SME provided expertise in the experimental design, analysis, and interpretation of the molecular markers’ expression levels. SHE actively participated in the coordination of the research and assisted in manuscript preparation. HMS contributed to data collection and analysis related to whole blood parameters. All authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
Distribution of mean catalase in different study groups (Group I–Group VIII). The definition of different groups is shown in Table 1.
Distribution of mean B-cell lymphoma-2 per different study groups (Group I–VIII). The definition of different groups is shown in Table 1.
Histopathological examination of testicular tissue (hematoxylin and eosin staining) of (a) Group I, (b) Group II, (c) Group III, (d) Group IV, (e) Group V, (f) Group VI, (g) Group VII, and (h) Group VIII in ×10 (left) and ×40 (right), respectively. The definition of different groups is shown in Table 1.
ACKNOWLEDGMENTS
This study was supported throughby funding from the Prince Sattam bin Abdulaziz University Project (No. PSAU/2023/R/1444). This research was funded by the Princess Nourah bint Abdulrahman University Researchers Supporting Project (No. PNURSP2023R99), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.
Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Distribution of mean catalase in different study groups (Group I–Group VIII). The definition of different groups is shown in Table 1.
Distribution of mean B-cell lymphoma-2 per different study groups (Group I–VIII). The definition of different groups is shown in Table 1.
Histopathological examination of testicular tissue (hematoxylin and eosin staining) of (a) Group I, (b) Group II, (c) Group III, (d) Group IV, (e) Group V, (f) Group VI, (g) Group VII, and (h) Group VIII in ×10 (left) and ×40 (right), respectively. The definition of different groups is shown in Table 1.