1. Introduction
The incidence of cancer has increased significantly throughout the world in recent years [ 1 ]. Formerly, surgery used to be the sole option for treating patients with solid tumors, which had a high fatality rate. Chemotherapy has improved such patient survival rates over the past 40 years [ 2 ]. Various chemotherapeutic agents, including cyclophosphamide (CP), are widely used as a part of their treatment regimen [ 3 ]. Unfortunately, it is non-selective for diseased cells [ 4 , 5 ]. CP has two active metabolites, namely, phosphoramide mustard and acrolein. While phosphoramide mustard has been associated with the antineoplastic and immunosuppressive actions of CP, acrolein is responsible for the toxic side effects of CP that include apoptosis and necrosis in normal tissue [ 6 ]. CP administration has been linked to significant side effects, such as hemorrhagic cystitis, gonadotoxicity, nephrotoxicity, and cardiotoxicity, limiting its therapeutic use. Moreover, female patients who are treated with CP are susceptible to early menopause. The risk of irreversible infertility increases significantly in both male and female patients when exposed to a cumulative medical dose [ 7 , 8 ]. CP has been reported to impact male fertility both centrally and peripherally via downregulating the hypothalamic–pituitary–gonadal (HPG) axis through affecting the hypothalamic Kiss1 mRNA expression and gonadotropin secretion, testicular steroidogenesis, testosterone synthesis, and eventually spermatogenesis [ 9 , 10 ]. Furthermore, Cp has been linked to direct testicular oxidative stress and DNA damage leading to testicular degeneration [ 11 , 12 ].
Flavonoids, found in many medicinal plants, fruit, and vegetables, can be useful in treating a variety of diseases. Flavonoids possess various pharmacological properties, including vasodilation, anti-allergic, immunostimulant, and antiviral effects [ 13 , 14 ]. They are reported to be effective as an antioxidant, anticarcinogen, antiproliferative, and in combating multidrug resistance, as well as in preventing chemotherapy-associated injury [ 15 ]. Hesperidin (HSP) is a bioflavonoid that is found mainly in citrus fruit such as oranges and lemons, as well as plant-derived liquids such as tea and olive oil. It has a wide range of pharmacological effects, including antioxidant, anti-inflammatory, anticarcinogenic, antiviral, antibacterial, antifungal, antiulcer, analgesic, and anticancer properties [ 16 ]. The main objective of the current work was to examine the potential therapeutic effect of HSP in preventing the progression of male infertility in rats treated with CP and its effects on the various molecular processes involved in male reproduction.
2. Results
2.1. Effect on the Final Body, Testicular Weights and Serum Hormone Levels
The final body and testicular weights of the CP-treated group were significantly lower ( p < 0.05) compared to the control group in Figure 1 A,B. The CP+HSP-treated group showed a significant increase in final body and testicular weights compared to the CP-treated group in Figure 1 A,B.
The serum levels of testosterone, FSH, LH, and prolactin showed a significant decrease in the CP-treated group in comparison to the control group ( p < 0.05) in Figure 1 C–F. The serum levels of testosterone, FSH, LH, and prolactin in the CP+HSP-treated group showed a significant increase compared to the CP-treated group, shown in Figure 1 C–F.
2.2. Effect on Testicular Lipid Peroxidation and Oxidative Stress Markers
CP-induced oxidative stress was indicated, with a significant decrease in GPx and TAC levels in the CP-treated group in comparison to that in the control group ( p < 0.05), shown in Figure 2 B,C. A significant increase in the MDA level, the lipid peroxidation marker, in the CP-treated group in comparison to that in the control group ( p < 0.05) is also shown in Figure 2 A. The CP+HSP-treated rats showed a significant decrease in testicular MDA levels, as well as a significant increase in testicular GPx and TAC levels, in comparison to those in the CP-treated group, shown in Figure 2 A–C.
2.3. Effect on Testicular Histopathology and Morphology
The control and the HSP groups showed normal morphology. The majority of seminiferous tubules were rounded in shape with a regular contour and few interstitial spaces containing clusters of interstitial cells. The lumina of most tubules revealed aggregated sperm bundles in Figure 3 A,B. A complete series of spermatogenic cells were seen (spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids). The seminiferous tubules were covered by a single layer of myoid cells with flattened nuclei. The interstitial spaces were narrow and contained normal Leydig cells and blood vessels, as shown in Figure 3 E,F.
Sections from CP-treated groups revealed degenerative testicular changes, including loss of their normal architecture, compared with the control group. Distorted seminiferous tubules with exfoliation of germ cells within their lumina and irregular outlines and germinal epithelium with some atrophic part. The spermatogenic cells appeared with darkly stained pyknotic nuclei and multinucleated giant cells were also seen. The interstitial tissues were wide and edematous. Numerous vacuolations were also seen, as shown in Figure 3 C,G.
Sections from CP+HSP-treated groups showed a marked improvement in the histological structure of testicular tissue in comparison to the CP-treated group. The majority of the seminiferous tubules almost regained their normal architecture, had nearly regular outlines, and were lined by stratified germinal epithelium. A few seminiferous tubules were lined by disorganized germinal epithelium separated from the underlying basement membrane. The lumina of most of them showed aggregations of sperms, while others were empty. Normal blood vessels and clusters of Leydig cells were also noticed in the relatively narrow interstitium with some vacuolations still observed, as shown in Figure 3 D,H.
Evaluation of Johnsen’s scores in histopathological sections indicated that there was a significant decrease in spermatogenesis quality in the CP-treated group compared to that in the control group ( p < 0.001). When compared with the CP-treated group, the CP+HSP-treated group showed a significant rise, but was still significantly different to the control group in Figure 3 I. Evaluation of the mean thickness of the capsule showed a significant increase in the CP-treated group in comparison to that of the control group ( p < 0.001). The CP+HSP-treated group showed a significant decline when compared with the CP-treated group, but was still significantly different from that in the control group, as shown in Figure 3 J. The CP-treated group’s mean thickness of the germinal epithelium was significantly decreased when compared to that of the control group ( p < 0.001). The CP+HSP-treated group showed a significant increase in the thickness of germinal epithelium when compared with the CP-treated group, as shown in Figure 3 K. The mean thickness of the interstitial space in the CP-treated group showed a significant increase in comparison to the control group ( p < 0.001). When compared to the CP-treated group, the CP+HSP-treated group showed a significant decline in Figure 3 L. The number of Leydig cells was significantly decreased in the CP-treated group compared to that in the control group ( p < 0.001). However, the CP+HSP-treated group showed a significant increase in comparison to the CP-treated group, as shown in Figure 3 M.
2.4. Effect on Testicular Immunohistochemistry of iNOS and P53
Examination of the iNOS immunohistochemical stained section of the control and HSP-treated group revealed mild expression of iNOS immune reactivity in the cytoplasm of the germ cells of seminiferous tubules, shown in Figure 4 A,B,E,F. The CP-treated group expressed a significant increase in iNOS immune reactivity in the cytoplasm of the germ cells and the interstitial cells, shown in Figure 4 C,G, whereas administration of HSP led to a marked decrease in iNOS immunoreactivity in the cytoplasm of the germ cells of seminiferous tubules and the interstitial cells in Figure 4 D,H. The mean area % of iNOS expression in the testes sections (X400) of the four experimental groups, was considerably higher in the CP-treated group in comparison to the control and CP+HSP-treated group, shown in Figure 4 M.
In the seminiferous tubules of the control group and HSP-treated group, the P53 immunopositive reaction was primarily found in the nucleus of apoptotic cells, and is hardly noticeable in Figure 4 I,J. Nevertheless, the seminiferous tubules of the CP-treated group had a large number of P53 immunopositive cells, shown in Figure 4 K; however, the seminiferous tubules of the CP+HSP-treated group contained fewer P53 immunopositive cells, shown in Figure 4 L. Regarding the average area % of P53 expression and the number of P53 immunopositive cells per tubule in the testes sections (X400) of the four experimental groups, the CP-treated group showed a significant increase compared to the control and CP+HSP-treated groups, shown in Figure 4 N,O. The CP+HSP-treated group showed a significant decrease in comparison to the CP-treated group, shown in Figure 4 N,O.
2.5. Effect on mRNA Expression of Hypothalamic KISS-1, KISS-1r, GnRH, Hypophyseal GnRHr and Testicular Steroidogenic Enzymes
A significant downregulation in hypothalamic KISS-1, KISS-1r, and GnRH and hypophyseal GnRHr mRNA expression in the CP-treated group in comparison to that in the control group were noticed. Treatment with HSP significantly upregulated hypothalamic KISS-1 and GnRH and hypophyseal GnRHr, but not KISS-1r, in comparison to that in the CP-treated group, as shown in Figure 5 A–D. In comparison to the control group, a significant downregulation in the testicular mRNA expression of StAR, CYP11A1, CYP17A1, HSD17B3, and CYP19A1 in the CP-treated group was noticed. In comparison to the CP-treated group, the CP+HSP-treated group showed considerable upregulation in the testicular StAR, CYP11A1, CYP17A1, HSD17B3, and CYP19A1, as shown in Figure 5 E–I.
2.6. Effect on Testicular mRNA Expression of GLP-1, PGC-1, and PPAR-α
A significant decrease in testicular GLP-1, PGC-1, and PPAR-a was shown in the CP-treated group compared to the control group in Figure 6 A–C. HSP administration showed a significant increase in testicular GLP-1, PGC-1, and PPAR-α when compared to that in the CP-treated group, shown in Figure 6 A–C.
2.7. Effect on Testicular Apoptotic and Inflammatory Marker
A significant upregulation in testicular P53 and IL1B mRNA expression and downregulation in testicular IL10 mRNA expression were detected in the CP-treated group when compared to that of the control group, as shown in Figure 7 A–C. The CP+HSP-treated group showed significant downregulation of testicular P53 and IL1B and upregulation in testicular IL10 mRNA expression when compared to CP-treated group, shown in Figure 7 A–C.
3. Discussion
Cancer is a leading cause of death worldwide [ 17 ]. As a result, researchers have been looking for different therapeutic strategies to improve the quality of life for patients. CP is a regularly used form of chemotherapy particularly in multiple myeloma, sarcoma, lymphoma, neuroblastoma, leukemia, and prostate and breast cancer [ 18 ]. CP has been shown to affect rapidly proliferating tissues such as gonads by interfering with their cell growth and differentiation [ 19 ]. CP treatment combined with several protective supplements, including polyphenols, vitamins, and minerals, has been suggested [ 20 ] to overcome such damaging impacts. HSP, owing to its antioxidant/anti-inflammatory effects, could be beneficial [ 21 , 22 ].
In the present work, the final body weight of rats receiving CP treatment was significantly decreased when compared with the control group, as previously reported [ 23 ]. A single intraperitoneal injection of a lesser dose of 120 mg/kg/b.wt [ 24 ] or 50 mg/kg/day for three days [ 25 ] also was reported to decrease body weight in rats. This decline could be caused by metabolic alterations causing appetite loss and decreased food intake. Yet, no weight change was noticed after administering CP in a single intraperitoneal dose of 200 mg/kg/b.wt. in rats [ 26 ]. HSP administration at a dose of 200 mg/kg/b.wt has been linked to improving the body weight in the aluminum phosphatide-treated group [ 27 , 28 ]. In the present study, the CP-treated group’s testicular weight significantly decreased when compared with the control group [ 29 ]. Administration of CP in a dose of 15 mg/kg once a week for 35 days has been linked to reduced testicular size in mice [ 30 ]. Other reports administering single CP at a dose of 100 mg/kg showed no difference in the weight of testis between groups [ 31 , 32 ]. HSP in a dose of 25–50 mg/kg for 60 days, revealed that HSP co-administration normalizes testicular weight [ 33 ]. Collectively, this could suggest a potential effect for the administration route, dose, treatment duration, and sensitivity of the animals in body and testicular weight changes.
The hypothalamic–pituitary–gonadal (HPG) axis controlling gonadal function in males starts by hypothalamic secretion of GnRH to stimulate the pituitary generation of FSH and LH. Additionally, FSH and LH play a role in regulating Leydig cell function to produce testosterone in males [ 8 ]. CP markedly reduced the serum levels of testosterone, FSH, LH, and prolactin [ 11 ], even at a single dose of 100 mg/kg [ 34 ]. The results of this study indicated that CP affected spermatogenesis by interfering with cellular processes as well as the pituitary–testicular axis. HSP significantly enhanced the serum testosterone, FSH, LH, and prolactin levels following bisphenol A administration [ 35 , 36 ]. Also, the administration of other flavonoids, including quercetin [ 11 ] or morin [ 37 , 38 ], or rutin [ 38 ], improved serum levels of testosterone, FSH, LH, and prolactin. This could be attributed to the protective roles of flavonoids in terms of anti-inflammatory and antioxidant ability.
CP disrupts the antioxidant defense mechanisms by producing large amounts of ROS in conjunction with its harmful metabolite, acrolein, which is known to increase oxidative stress, causing a marked reduction in GPx and TAC levels and a marked elevation in MDA levels [ 39 , 40 ]. Oxidative stress is a major contributor to male infertility [ 41 ] via modifications in microvascular blood flow leading to elevated rates of germ cell death. HSP co-administration reduced the level of the lipid peroxidation marker, MDA, and also strengthened the body’s natural antioxidant defense system by boosting the activity of antioxidant enzymes such as SOD and GPx [ 27 , 42 , 43 , 44 ]. Oxidative stress, lipid peroxidation, and inflammation usually manifest histological and functional abnormalities in testicular tissue [ 45 , 46 ]. CP intraperitoneal administration at a dose of 5 mg/kg daily for four weeks, showed intraepithelial vacuoles as an indication of seminiferous tubules atrophy, as well as spacing and separation of germ cells, which may be caused by sloughing and exfoliation of the germ cells due to the primary impact on the cell-to-cell junction in between Sertoli cells and germ cells [ 47 , 48 ].
Other reports of single CP administration at a dose of 200 mg/kg showed marked testicular damage, including interstitial bleeding, and separation of spermatogenic cells with the presence of vacuoles, and explained how CP could cause oxidative stress, lipid peroxidation, and apoptosis [ 49 ]. CP intraperitoneal administration at a dose of 60 mg/kg per week for eight weeks revealed that the seminiferous tubules exhibited morphological changes, including interstitial edema, vacuolization, multinucleated giant cell development, desquamation, degeneration, and disorganization. In addition, reactive oxygen species and oxidative stress have been linked to the pathogenesis of CP toxicity [ 50 ].
The CP-treated group revealed a significant increase in iNOS, only formed during inflammation, ischemia, and apoptosis [ 51 ], immune reactivity in the cytoplasm of the cells of the seminiferous tubules [ 52 ]. Exposure to cisplatin, aluminum chloride, methotrexate, and silica nanoparticles reported an increase in testicular iNOS expression [ 53 , 54 , 55 , 56 ]. Anticancer drugs, including cyclophosphamide, methotrexate, tamoxifen, doxorubicin, and 5-fluorouracil administration, have been associated with the induction of oxidative stress and inflammation [ 57 ]. Such results, confirmed by another flavonoid (quercetin), [ 54 ] found that coadministration of quercetin to the aluminum chloride-treated group decreased the iNOS expression in testis tissue. HSP caused a marked decrease in the CP-induced expression of iNOS in the liver [ 58 ] and renal tissue [ 59 ]. Coadministration of another antioxidant as melatonin decreased the expression of iNOS in testicular tissue in the cisplatin-treated group [ 60 ].
Increased testicular apoptosis, as indicated by increased P53 expression, results in spermatogenic arrest and a reduction in spermatogonia, spermatocytes, and spermatid numbers [ 61 ]. The CP-treated group revealed a marked increase in P53 immune reactivity in the nucleus of the cells of the seminiferous tubules and revealed an increased number of apoptotic cells in the CP-treated group [ 62 ]. Exposure to potassium dichromate, D-gal/NaNO 2 , bleomycin, etoposide, and cisplatin resulted in an increase in testicular P53 expression [ 63 , 64 , 65 ]. Treatment with hesperidin might protect mice’s testis against apoptosis [ 66 ]. HSP administration at a dose of 50 mg/kg daily via gavage for 14 days, reported mitigated testicular alterations brought on by cisplatin [ 67 ]. Additionally, HSP treatment reduced the testicular seminiferous tubules deterioration process and lowered ischemia/reperfusion-induced reproductive damage [ 68 ]. HSP caused a marked decrease in the expression of P53 in the corneal tissue [ 69 ], liver [ 22 ], and colon [ 70 ]. Regarding other flavonoids, co-administration of quercetin to the cisplatin-treated group decreased the expression of P53 in testicular tissue [ 71 ].
The mean testicular Johnsen’s score in the CP-treated group decreased significantly in comparison with the control group [ 52 ]. The thickness of germinal epithelium had significantly decreased and the lumina were large and empty [ 72 ]. They linked these findings to the inhibition of B-spermatogonia mitosis, which denotes an extension of the G1 phase of the cell cycle growth. Additionally, it has been demonstrated that this epithelial thinning results in a deficiency in sperm production. A significant decline in epithelial height and increase in the interstitial space in the cisplatin-treated group [ 73 ] could be attributed to oxidative stress that was responsible for all cisplatin-induced damage in the testis. The thickness of the capsule of the testis was significantly increased in the iprodione-treated group [ 35 ]. The number of Leydig cells decreased significantly in the CP-treated group [ 74 , 75 ]. In the present study, there was a significant improvement in testicular Johnsen’s score in the CP+HSP-treated group when compared to the CP-treated group. This result was consistent with previous reports indicating that diabetic rats treated with HSP showed considerable improvement in the mean testicular biopsy score [ 76 ].
In the present study, the number of Leydig cells in the CP+HSP-treated group increased significantly when compared to the CP-treated group [ 77 ]. HSP treatment with cisplatin causes considerable improvement in the thickness of germinal epithelium [ 67 ]. Quercetin co-treatment showed a significant reduction in the interstitial space in the arsenic-treated group [ 78 ]. Melatonin co-treatment showed a significant reduction in the thickness of tunica albuginea in the taxol-treated group [ 79 ].
In this study, there was significant downregulation of hypothalamic KISS, KISSr, GnRH, and hypophyseal GnRHr in the CP-treated group when compared with the control group. Oxidative stress induced by extensive exercise has been reported to downregulate the expression of KISS, KISSr, GnRH, and GnRHr [ 80 ]. A significant upregulation in gene expression of hypothalamic KISS, GnRH, and hypophyseal GnRHr in the CP+HSP-treated group if compared with the CP-treated group was noticed. This could be attributed to the flavonoid’s ability to prevent tissue damage, prevent the inactivation of steroidogenesis, and increase gonadotropin release [ 81 , 82 ]. CP treatment significantly decreases steroidogenic genes that include StAR, CYP11A1, CYP17A1, HSD17B, and CYP1719A1 compared with other groups [ 11 , 83 ]. Cisplatin has been reported to induce similar impacts [ 84 , 85 ]. HSP administration enhances testicular functions through the upregulation of steroidogenesis-related genes [ 86 ]. Additionally, isorhamnetin, a bioflavonoid treatment, significantly improved testosterone production through the upregulation of steroidogenic genes and antioxidant ability [ 87 ]. On the other hand, studies have also suggested that flavonoids may interact with estrogen receptors (ERs) to modulate the activity of the endocrine system [ 88 , 89 ]. This interaction has been linked to a variety of health outcomes, including testicular dysfunction in rats [ 88 ]. The interaction between flavonoids and ERs is complex and depends on the type of flavonoid, duration of administration and its concentration. Such interaction should be in focus in future research.
GLP-1 is essential for maintaining male fertility, since it controls spermatogenesis and steroidogenesis directly. It is released by Leydig cells and works on Sertoli, germinal epithelial, and Leydig cells (all of which have GLP-1 receptors). This procedure increases the metabolism of Sertoli cells and raises sperm cell quality [ 90 ]. PPAR-α and PGC-1α, stimulate fatty acid oxidation and impact the testicular energy balance [ 91 ]. Testicular torsion, similar to CP administration, caused significant downregulation of testicular GLP-1, PPAR-α, and PGC-1α and affect the metabolism of testicular cells and worsens oxidative stress by raising reactive oxygen species and reactive nitrogen species, which promote apoptosis and inflammation inside testis [ 92 ]. A significant upregulation of testicular GLP-1, PPAR-α, and PGC-1α in the CP+HSP-treated group was noticed, indicating a positive effect on testicular metabolism. CP-treated rats showed a significant upregulation of testicular apoptotic P53 [ 83 ] and pro-inflammatory IL1β and downregulation of anti-inflammatory IL10. Cisplatin caused a significant elevation in pro-inflammatory IL1β and significant downregulation of the anti-inflammatory IL10 gene respectively [ 84 , 93 ]. HSP’s antioxidant properties cleared the ROS, preventing the pro-inflammatory genes from activating, and protecting testicular tissue from inflammation [ 94 ]. CP and HSP-combined treatment downregulated the testicular apoptotic P53 gene and pro-inflammatory IL1β and significant upregulated the anti-inflammatory IL10 gene when compared with CP-treated group. Co-treatment with bilobetin, a natural bioflavonoid, significantly reduced testicular P53 expression and significantly increased testicular IL10 expression in the cisplatin-treated group [ 95 ].
4. Materials and Methods
4.1. Chemicals
Cyclophosphamide in the form of powder acquired from Baxter oncology GmbH in Germany. CP was injected at a dose of 150 mg/kg/B.wt. Hesperidin (>80% purity powder CAS NO 520-26-3) was produced by Sigma-Aldrich Company St. Louis, MO, USA, and acquired from Sigma-Egypt. Hesperidin was orally administrated by gavage at a dose of 200 mg/kg/B.wt.
4.2. Experimental Animals
Thirty-two adult healthy male Sprague Dawley rats (12–14 weeks) weighing 210 ± 10 g were obtained from Zagazig scientific and medical research center (ZSMRC). The animals were then kept at a constant 23 ± 2 °C and operated on a 12-h light/12-h dark cycle. Throughout the study, the animals were kept on a regular diet and ad libitum water supply. All rats received humane care and the experimental methods were approved by the Institutional Animal Care and Use Committee of Zagazig University (No. ZU-IACUC/3/F/172/2019).
4.3. Experimental Design and Sample Collection
The rats were randomly assigned into four main groups (n = 8), namely, the control group that received normal saline for eight days, the HSP-treated group that received HSP 200 mg/kg/d orally for eight days [ 27 , 96 ], the CP-treated group that received CP 150 mg/kg single intraperitoneal injection on the 1st day of the experiment [ 87 ], and CP+HSP-treated group that received CP 150 mg/kg single intraperitoneal injection on the 1st day of the experiment and HSP 200 mg/kg/d orally for eight days. Rats were euthanized 48 h after the last HSP dose. The body weights were determined and venous blood samples were taken from their retro-orbital plexus using a capillary glass tube, blood was left to clot at room temperature, then centrifuged at 3000 rpm for 10 min to separate the serum. The samples were then kept at −20 °C to be used subsequently for hormonal assay estimation. Then, the rats were anesthetized by intraperitoneal injection of thiopental (75 mg/kg BW) and subjected to cervical dislocation, the abdomen was then opened to collect both testes outside the body, weighted and then divided into three parts; the first part was collected on 10% neutral buffered formalin for histopathological and immunohistochemical examination, the second part (30 mg) was removed directly on liquid nitrogen and then kept at −80 °C to be used for total RNA extraction and the third part (1 g) was homogenized to be used for different biochemical tests. After being dissected, the hypothalamus and pituitary gland were stored in liquid nitrogen and kept there at −80 °C until total RNA extraction as previously described [ 97 ].
4.4. Hormonal and Biochemical Analysis
The levels of serum of FSH, LH, total testosterone, and prolactin were determined by using a commercially available rat enzyme-linked immunosorbent assay (ELISA) (Catalog No. CSB-E12654r) for LH as well as (Catalog No. CSB-E06869r) for FSH and (Catalog No. MBS282195) for testosterone and (Catalog No. CSB-E06881r) for prolactin [ 98 , 99 ]. The levels of malondialdehyde (MDA), glutathione peroxidase enzyme activity (GPx), and total antioxidant capacity (TAC) were assessed using (Catalog No. (ELA-E0597r) for MDA, (Catalog No. ELA-E0295r) for GPx, (Catalog No. STA-360) for TAC.
4.5. Real-Time Quantitative RT-PCR (qRT-PCR) Analysis
Briefly, total RNA was extracted using Trizol (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA), and for evaluating the RNA quality, the A260/A280 ratio was analyzed using the NanoDrop VR ND-1000 Spectrophotometer (NanoDrop Technologies; Wilmington, DE, USA). For cDNA synthesis, a High-Capacity cDNA Reverse Transcription Kit cDNA Kit (Applied Biosystems™, USA) was used, followed by the preparation of the primers according to their manufacturer instructions, Sangon Biotech (Beijing, China), as provided in Table 1 .
Real-time RT-PCR was performed in Mx3005P real-time PCR system (Agilent Stratagene, USA) using TOPrealTM qPCR 2X PreMIX (SYBR Green with low ROX) (Cat. # P725 or P750) (Enzynomics, Korea) following the manufacturer’s instructions. The PCR cycling conditions included initial denaturation at 95 °C for 12 min followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. A melting curve analysis was performed following PCR amplification. The expression level of the target genes was normalized using the mRNA expression of a known housekeeping gene, Gapdh. Results are expressed as fold-changes compared to the control group following the 2 −ΔΔCt method [ 100 ].
4.6. Histopathological and Immunohistochemical Examination
All the testicular specimens from all groups were installed in Bouin solution for 4 to 5 h till converted to a hard consistency then kept for paraffin block preparation. For the light microscopic analysis, tissue sections of 5 μm thickness were stained with H and E stain to examine the structural light microscopic alterations [ 101 ]. For immunohistochemical staining, a rabbit monoclonal antibody of IgG type was designed for specific localization of inducible nitric oxide synthase (iNOS) marker for oxidative stress and P53 (apoptosis marker) in paraffin sections. The kits were delivered from DAKO life trade Egypt (Catalog No. A0312 for iNOS and Catalog No. A5761 for P53). Following the manufacturer’s recommendations, sections were inspected and photographed by light microscope LEICA DM500 in the Anatomy Department, Faculty of Medicine, Zagazig University
The Johnsen scoring system was used to assess the histological alterations in testicular tissue. Each tubule received a Johnsen’s score between 1 (extremely poor) and 10 (excellent) following Johnsen’s criteria [ 102 ]. Image J analysis software (Fiji image j; 1.51 n, NIH, USA) was used for measuring the thickness of the capsule, the thickness of the germinal epithelium, the thickness of the interstitial space, and the count of Leydig cells in sections stained with H&E per 100 high powers fields for the thickness of capsule and 400 high powers fields for the thickness of the germinal epithelium, the thickness of the interstitial space and count of Leydig cells [ 103 ]. After immunostaining, the number of apoptotic cells (in P53 stained sections) was counted and analysis of the mean area % of P53 (in P53 stained sections) or iNOS (in iNOS stained sections) was performed in nearly 150 seminiferous tubules from 8 animals/group, and the findings were statistically analyzed [ 104 ].
4.7. Statistical Analysis
Continuous variables were represented by the mean ± standard error mean (SEM). All data were normally distributed and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference test in homogenous data for multiple group comparison. p values less than 0.05 ( p < 0.05) was statistically significant using the statistical software package SPSS for Windows (Version 20; SPSS Inc., Chicago, IL, USA).
5. Conclusions
Our study concludes that hesperidin’s antioxidant, anti-inflammatory, and anti-apoptotic activities could modulate testicular disturbances induced by cyclophosphamide. The findings of our study raise the prospect of hesperidin, a bioflavonoid, as a therapeutic intervention for delaying testicular dysfunction. Such findings could be an important entry point for preserving fertility while using antineoplastic drugs such as cyclophosphamide against a wide spectrum of malignancies. Although the therapeutic applications of bioflavonoids, including hesperidin, should be promising, any future clinical application should be preceded by rigorous clinical studies.
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Author Contributions
Conceptualization, A.A.H., E.R.A., S.S.A.E.-F., T.K. and M.M.M.A.; methodology, A.A.H., E.R.A., S.S.A.E.-F., T.K., L.M.F. and A.H.A.; software, T.K., L.M.F. and A.H.A.; validation, A.A.H., E.R.A., S.S.A.E.-F., T.K., L.M.F., A.H.A. and M.M.M.A.; formal analysis, S.S.A.E.-F., T.K., L.M.F., A.H.A. and M.M.M.A.; investigation, A.A.H., E.R.A., S.S.A.E.-F., T.K., L.M.F., A.H.A. and M.M.M.A.; resources, S.S.A.E.-F., T.K., L.M.F. and A.H.A.; data curation, S.S.A.E.-F., T.K. and A.H.A.; writing—original draft preparation, S.S.A.E.-F., T.K. and A.H.A.; writing—review and editing, A.A.H., E.R.A., S.S.A.E.-F., T.K., L.M.F., A.H.A. and M.M.M.A.; visualization, supervision, project administration, A.A.H., E.R.A., T.K., L.M.F., A.H.A. and M.M.M.A.; funding acquisition, S.S.A.E.-F., M.M.M.A., T.K., L.M.F. and A.H.A. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
All rats received humane care and the experimental methods were approved by the Institutional Animal Care and Use Committee of Zagazig University (No. ZU-IACUC/3/F/172/2019).
Informed Consent Statement
Not applicable.
Data Availability Statement
The data presented in this study are available upon reasonable request from the corresponding author. The data are not publicly available as it contains information that could compromise the privacy of an ongoing research.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Yilmaz E. Coskun E.I. Sahin N. Ciplak B. Ekici K. MPV, NLR, and platelet count: New hematologic markers in diagnosis of malignant ovarian tumor Eur. J. Gynaecol. Oncol. 2017 38 346 349 29693870
2. Klareskog L. van der Heijde D. de Jager J.P. Gough A. Kalden J. Malaise M. Mola E.M. Pavelka K. Sany J. Settas L. Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: Double-blind randomised controlled trial Lancet 2004 363 675 681 10.1016/S0140-6736(04)15640-7 15001324
3. Veal G.J. Cole M. Chinnaswamy G. Sludden J. Jamieson D. Errington J. Malik G. Hill C.R. Chamberlain T. Boddy A.V. Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin’s lymphoma Eur. J. Cancer 2016 55 56 64 10.1016/j.ejca.2015.12.007 26773420 PMC4778608
4. Boisseaux P. Noury P. Thomas H. Garric J. Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A Ecotoxicol. Environ. Saf. 2017 139 358 366 10.1016/j.ecoenv.2017.02.003 28189777
5. Grzesiuk M. Mielecki D. Pilżys T. Garbicz D. Marcinkowski M. Grzesiuk E. How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome PLoS ONE 2018 13 e0195366 10.1371/journal.pone.0195366 29621334 PMC5886542
6. Temel Y. Çağlayan C. Ahmed B.M. Kandemir F.M. Çiftci M. The effects of chrysin and naringin on cyclophosphamide-induced erythrocyte damage in rats: Biochemical evaluation of some enzyme activities in vivo and in vitro Naunyn Schmiedebergs Arch Pharm. 2021 394 645 654 10.1007/s00210-020-01987-y 33104847
7. Spears N. Lopes F. Stefansdottir A. Rossi V. De Felici M. Anderson R.A. Klinger F.G. Ovarian damage from chemotherapy and current approaches to its protection Hum. Reprod. Update 2019 25 673 693 10.1093/humupd/dmz027 31600388 PMC6847836
8. Akomolafe S.F. Aluko B.T. Protective effect of curcumin on fertility in cyclophosphamide exposed rats: Involvement of multiple pathways J. Food Biochem. 2020 44 e13095 10.1111/jfbc.13095 31709586
9. Khamis T. Abdelalim A.F. Saeed A.A. Edress N.M. Nafea A. Ebian H.F. Algendy R. Hendawy D.M. Arisha A.H. Abdallah S.H. Breast milk MSCs upregulated β-cells PDX1, Ngn3, and PCNA expression via remodeling ER stress/inflammatory/apoptotic signaling pathways in type 1 diabetic rats Eur. J. Pharmacol. 2021 905 174188 10.1016/j.ejphar.2021.174188 34004210
10. Khamis T. Abdelalim A.F. Abdallah S.H. Saeed A.A. Edress N.M. Arisha A.H. Early intervention with breast milk mesenchymal stem cells attenuates the development of diabetic-induced testicular dysfunction via hypothalamic Kisspeptin/Kiss1r-GnRH/GnIH system in male rats Biochim. Et Biophys. Acta-Mol. Basis Dis. 2020 1866 165577 10.1016/j.bbadis.2019.165577 31672553
11. Ebokaiwe A.P. Obasi D.O. Njoku R.C. Osawe S. Cyclophosphamide-induced testicular oxidative-inflammatory injury is accompanied by altered immunosuppressive indoleamine 2, 3-dioxygenase in Wister rats: Influence of dietary quercetin Andrologia 2022 54 e14341 10.1111/and.14341 34854117
12. Van den Boogaard W.M.C. Komninos D.S.J. Vermeij W.P. Chemotherapy Side-Effects: Not All DNA Damage Is Equal Cancers 2022 14 627 10.3390/cancers14030627 35158895 PMC8833520
13. Naz S. Imran M. Rauf A. Orhan I.E. Shariati M.A. Iahtisham Ul H. IqraYasmin Shahbaz M. Qaisrani T.B. Shah Z.A. Chrysin: Pharmacological and therapeutic properties Life Sci. 2019 235 116797 10.1016/j.lfs.2019.116797 31472146
14. Gulcin İ. Antioxidants and antioxidant methods: An updated overview Arch. Toxicol. 2020 94 651 715 10.1007/s00204-020-02689-3 32180036
15. Kopustinskiene D.M. Jakstas V. Savickas A. Bernatoniene J. Flavonoids as Anticancer Agents Nutrients 2020 12 457 10.3390/nu12020457 32059369 PMC7071196
16. Shoorei H. Banimohammad M. Kebria M.M. Afshar M. Taheri M.M. Shokoohi M. Farashah M.S. Eftekharzadeh M. Akhiani O. Gaspar R. Hesperidin improves the follicular development in 3D culture of isolated preantral ovarian follicles of mice Exp. Biol. Med. 2019 244 352 361 10.1177/1535370219831615 PMC6488865 30781997
17. Lehmann V. Chemaitilly W. Lu L. Green D.M. Kutteh W.H. Brinkman T.M. Srivastava D.K. Robison L.L. Hudson M.M. Klosky J.L. Gonadal Functioning and Perceptions of Infertility Risk Among Adult Survivors of Childhood Cancer: A Report From the St Jude Lifetime Cohort Study J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2019 37 893 902 10.1200/JCO.18.00965 PMC6455717 30811296
18. Fusco R. Salinaro A.T. Siracusa R. D’Amico R. Impellizzeri D. Scuto M. Ontario M.L. Crea R. Cordaro M. Cuzzocrea S. Hidrox( ® ) Counteracts Cyclophosphamide-Induced Male Infertility through NRF2 Pathways in a Mouse Model Antioxidants 2021 10 778 10.3390/antiox10050778 34068924 PMC8156985
19. Ghobadi E. Moloudizargari M. Asghari M.H. Abdollahi M. The mechanisms of cyclophosphamide-induced testicular toxicity and the protective agents Expert Opin. Drug Metab. Toxicol. 2017 13 525 536 10.1080/17425255.2017.1277205 28019118
20. Yasueda A. Urushima H. Ito T. Efficacy and Interaction of Antioxidant Supplements as Adjuvant Therapy in Cancer Treatment: A Systematic Review Integr. Cancer Ther. 2016 15 17 39 10.1177/1534735415610427 26503419 PMC5736082
21. Zouchoune B. How the ascorbic acid and hesperidin do improve the biological activities of the cinnamon: Theoretical investigation Struct. Chem. 2020 31 2333 2340 10.1007/s11224-020-01594-w 32837117 PMC7361000
22. Afzal S.M. Vafa A. Rashid S. Barnwal P. Shahid A. Shree A. Islam J. Ali N. Sultana S. Protective effect of hesperidin against N,N’-dimethylhydrazine induced oxidative stress, inflammation, and apoptotic response in the colon of Wistar rats Environ. Toxicol. 2020 36 642 653 10.1002/tox.23068 33289288
23. Khorwal G. Chauhan R. Nagar M. Khorwal G. Effect of cyclophosphamide on liver in albino rats: A comparative dose dependent histomorphological study Int. J. Biomed. Adv. Res. 2017 8 102 107 10.7439/ijbar.v8i3.3953
24. Yao Y. Xu Y. Wang Y. Protective roles and mechanisms of rosmarinic acid in cyclophosphamide-induced premature ovarian failure J. Biochem. Mol. Toxicol. 2020 34 e22591 10.1002/jbt.22591 32711407
25. Omole J.G. Ayoka O.A. Alabi Q.K. Adefisayo M.A. Asafa M.A. Olubunmi B.O. Fadeyi B.A. Protective Effect of Kolaviron on Cyclophosphamide-Induced Cardiac Toxicity in Rats J. Evid.-Based Integr. Med. 2018 23 2156587218757649 10.1177/2156587218757649 29468886 PMC5871040
26. Razak R. Ismail F. Isa M.L.M. Wahab A.Y.A. Muhammad H. Ramli R. Ismail R. Ameliorative Effects of Aquilaria malaccensis Leaves Aqueous Extract on Reproductive Toxicity Induced by Cyclophosphamide in Male Rats Malays. J. Med. Sci.: MJMS 2019 26 44 57 10.21315/mjms2019.26.1.4 30914892 PMC6419872
27. Afolabi O.K. Wusu A.D. Ugbaja R. Fatoki J.O. Aluminium phosphide-induced testicular toxicity through oxidative stress in Wistar rats: Ameliorative role of hesperidin Toxicol. Res. Appl. 2018 2 2397847318812794 10.1177/2397847318812794
28. Hamdy S.M. Sayed O.N. Abdel Latif A.K.M. Abd-Elazeez A.M. Amin A.M. Protective Effect Of Hesperidin And Tiger Nut Against DMBA Carcinogenicity In Female Rats Biochem. Lett. 2016 12 150 167 10.21608/blj.2016.48192
29. Rezaei S. Hosseinimehr S.J. Zargari M. Karimpour Malekshah A. Mirzaei M. Talebpour Amiri F. Protective effects of sinapic acid against cyclophosphamide-induced testicular toxicity via inhibiting oxidative stress, caspase-3 and NF-kB activity in BALB/c mice Andrologia 2021 53 e14196 10.1111/and.14196 34333791
30. Mehraban Z. Ghaffari Novin M. Golmohammadi M.G. Sagha M. Ziai S.A. Abdollahifar M.A. Nazarian H. Protective Effect of Gallic Acid on Testicular Tissue, Sperm Parameters, and DNA Fragmentation against Toxicity Induced by Cyclophosphamide in Adult NMRI Mice Urol. J. 2020 17 78 85 10.22037/uj.v0i0.4858 30882156
31. Mohammadi F. Nikzad H. Taghizadeh M. Taherian A. Azami-Tameh A. Hosseini S.M. Moravveji A. Protective effect of Zingiber officinale extract on rat testis after cyclophosphamide treatment Andrologia 2014 46 680 686 10.1111/and.12135 23889539
32. Xie R. Chen L. Wu H. Chen T. Wang F. Chen X. Sun H. Li X. GnRH Antagonist Improves Pubertal Cyclophosphamide-Induced Long-Term Testicular Injury in Adult Rats Int. J. Endocrinol. 2018 2018 4272575 10.1155/2018/4272575 29983710 PMC6015689
33. Helmy H.S. Senousy M.A. El-Sahar A.E. Sayed R.H. Saad M.A. Elbaz E.M. Aberrations of miR-126-3p, miR-181a and sirtuin1 network mediate Di-(2-ethylhexyl) phthalate-induced testicular damage in rats: The protective role of hesperidin Toxicology 2020 433–434 152406 10.1016/j.tox.2020.152406 32050098
34. Shabaan S. Madi N. Elgharib M. Nasif E. Study the Effect of Silymarin on Cyclophosphamide Induced Testicular Damage in Adult Albino Rats Bull. Egypt. Soc. Physiol. Sci. 2021 41 553 564 10.21608/besps.2021.66777.1097
35. Hassan M.A. El Bohy K.M. El Sharkawy N.I. Imam T.S. El-Metwally A.E. Hamed Arisha A. Mohammed H.A. Abd-Elhakim Y.M. Iprodione and chlorpyrifos induce testicular damage, oxidative stress, apoptosis and suppression of steroidogenic- and spermatogenic-related genes in immature male albino rats Andrologia 2021 53 e13978 10.1111/and.13978 33586219
36. Kasem S.E. Abdelnaby A.A. Mohammed P.A. Hemdan S.B. Abd El-Fattah R.M.Z. Elsayed R.M. Protective Effect of Hesperidin on Kidneys and Testes of Adult Male Rats Exposed to Bisphenol A Egypt. J. Hosp. Med. 2022 88 3005 3013 10.21608/ejhm.2022.244529
37. Arisha A.H. Ahmed M.M. Kamel M.A. Attia Y.A. Hussein M.M.A. Morin ameliorates the testicular apoptosis, oxidative stress, and impact on blood–testis barrier induced by photo-extracellularly synthesized silver nanoparticles Environ. Sci. Pollut. Res. 2019 26 28749 28762 10.1007/s11356-019-06066-1 31376127
38. Hussein M.M.A. Gad E. Ahmed M.M. Arisha A.H. Mahdy H.F. Swelum A.A.A. Tukur H.A. Saadeldin I.M. Amelioration of titanium dioxide nanoparticle reprotoxicity by the antioxidants morin and rutin Environ. Sci. Pollut. Res. 2019 26 29074 29084 10.1007/s11356-019-06091-0 31392614
39. Salimnejad R. Soleimani Rad J. Nejad D. Protective Effect of Ghrelin on Oxidative Stress and Tissue Damages of Mice Testes Followed By Chemotherapy With Cyclophosphamide Crescent J. Med. Biol. Sci. 2018 5 138 143
40. Ekeleme-Egedigwe C.A. Famurewa A.C. David E.E. Eleazu C.O. Egedigwe U.O. Antioxidant potential of garlic oil supplementation prevents cyclophosphamide-induced oxidative testicular damage and endocrine depletion in rats J. Nutr. Intermed. Metab. 2019 18 100109 10.1016/j.jnim.2020.100109
41. Alkhalaf M.I. Alansari W.S. Alshubaily F.A. Alnajeebi A.M. Eskandrani A.A. Tashkandi M.A. Babteen N.A. Chemoprotective effects of inositol hexaphosphate against cyclophosphamide-induced testicular damage in rats Sci. Rep. 2020 10 12599 10.1038/s41598-020-68608-9 32724173 PMC7387554
42. Shokoohi M. Khaki A. Shoorei H. Khaki A.A. Moghimian M. Abtahi-Eivary S.H. Hesperidin attenuated apoptotic-related genes in testicle of a male rat model of varicocoele Andrology 2020 8 249 258 10.1111/andr.12681 31325243
43. Saber T.M. Arisha A.H. Abo-Elmaaty A.M.A. Abdelgawad F.E. Metwally M.M.M. Saber T. Mansour M.F. Thymol alleviates imidacloprid-induced testicular toxicity by modulating oxidative stress and expression of steroidogenesis and apoptosis-related genes in adult male rats Ecotoxicol. Environ. Saf. 2021 221 112435 10.1016/j.ecoenv.2021.112435 34171690
44. Saber T.M. Mansour M.F. Abdelaziz A.S. Mohamed R.M.S. Fouad R.A. Arisha A.H. Argan oil ameliorates sodium fluoride-induced renal damage via inhibiting oxidative damage, inflammation, and intermediate filament protein expression in male rats Environ. Sci. Pollut. Res. Int. 2020 27 30426 30436 10.1007/s11356-020-09366-z 32462624
45. Iqubal A. Syed M.A. Najmi A.K. Ali J. Haque S.E. Ameliorative effect of nerolidol on cyclophosphamide-induced gonadal toxicity in Swiss Albino mice: Biochemical-, histological- and immunohistochemical-based evidences Andrologia 2020 52 e13535 10.1111/and.13535 32048763
46. Hamzeh M. Hosseinimehr S.J. Karimpour A. Mohammadi H.R. Khalatbary A.R. Talebpour Amiri F. Cerium Oxide Nanoparticles Protect Cyclophosphamide-induced Testicular Toxicity in Mice Int. J. Prev. Med. 2019 10 5 10.4103/ijpvm.IJPVM_184_18 30774839 PMC6360849
47. Anan H.H. Zidan R.A. Abd El-Baset S.A. Ali M.M. Ameliorative effect of zinc oxide nanoparticles on cyclophosphamide induced testicular injury in adult rat Tissue Cell 2018 54 80 93 10.1016/j.tice.2018.08.006 30309514
48. Afkhami-Ardakani M. Hasanzadeh S. Shahrooz R. Delirezh N. Malekinejad H. Antioxidant effects of Spirulina platensis (Arthrospira platensis) on cyclophosphamide-induced testicular injury in rats Vet. Res. Forum: Int. Q. J. 2018 9 35 41 PMC5913559 29719662
49. Cengiz M. Sahinturk V. Yildiz S.C. Şahin İ.K. Bilici N. Yaman S.O. Altuner Y. Appak-Baskoy S. Ayhanci A. Cyclophosphamide induced oxidative stress, lipid per oxidation, apoptosis and histopathological changes in rats: Protective role of boron J. Trace Elem. Med. Biol. Organ Soc. Miner. Trace Elem. (GMS) 2020 62 126574 10.1016/j.jtemb.2020.126574 32516632
50. Torabi F. Malekzadeh Shafaroudi M. Rezaei N. Combined protective effect of zinc oxide nanoparticles and melatonin on cyclophosphamide-induced toxicity in testicular histology and sperm parameters in adult Wistar rats Int. J. Reprod. Biomed. 2017 15 403 412 10.29252/ijrm.15.7.403 29202124 PMC5601931
51. Nakazawa H. Chang K. Shinozaki S. Yasukawa T. Ishimaru K. Yasuhara S. Yu Y.M. Martyn J.A. Tompkins R.G. Shimokado K. iNOS as a Driver of Inflammation and Apoptosis in Mouse Skeletal Muscle after Burn Injury: Possible Involvement of Sirt1 S-Nitrosylation-Mediated Acetylation of p65 NF-κB and p53 PLoS ONE 2017 12 e0170391 10.1371/journal.pone.0170391 28099528 PMC5242494
52. Salama R.M. Abd Elwahab A.H. Abd-Elgalil M.M. Elmongy N.F. Schaalan M.F. LCZ696 (sacubitril/valsartan) protects against cyclophosphamide-induced testicular toxicity in rats: Role of neprilysin inhibition and lncRNA TUG1 in ameliorating apoptosis Toxicology 2020 437 152439 10.1016/j.tox.2020.152439 32197949
53. Hamza A.A. Elwy H.M. Badawi A.M. Fenugreek seed extract attenuates cisplatin-induced testicular damage in Wistar rats Andrologia 2016 48 211 221 10.1111/and.12435 25996647
54. Olanrewaju J.A. Akinpade T.G. Olatunji S.Y. Owolabi J.O. Enya J.I. Adelodun S.T. Fabiyi S.O. Desalu A.B. Observable Protective Activities of Quercetin on Aluminum Chloride-Induced Testicular Toxicity in Adult Male Wistar Rat J. Hum. Reprod. Sci. 2021 14 113 120 10.4103/jhrs.jhrs_190_20 34316225 PMC8279060
55. Sayed A.M. Hassanein E.H.M. Ali F.E.M. Omar Z.M.M. Rashwan E.K. Mohammedsaleh Z.M. Abd El-Ghafar O.A.M. Regulation of Keap-1/Nrf2/AKT and iNOS/NF-κB/TLR4 signals by apocynin abrogated methotrexate-induced testicular toxicity: Mechanistic insights and computational pharmacological analysis Life Sci. 2021 284 119911 10.1016/j.lfs.2021.119911 34450167
56. Azouz R.A. Korany R.M.S. Noshy P.A. Silica Nanoparticle-Induced Reproductive Toxicity in Male Albino Rats via Testicular Apoptosis and Oxidative Stress Biol. Trace Elem. Res. 2022 10.1007/s12011-022-03280-w PMC9931864 35672543
57. Famurewa A.C. Ekeleme-Egedigwe C.A. Onwe C.S. Egedigwe U.O. Okoro C.O. Egedigwe U.J. Asogwa N.T. Ginger juice prevents cisplatin-induced oxidative stress, endocrine imbalance and NO/iNOS/NF-κB signalling via modulating testicular redox-inflammatory mechanism in rats Andrologia 2020 52 e13786 10.1111/and.13786 32777091
58. Fouad A. Albuali W. Jresat I. Protective Effect of Hesperidin against Cyclophosphamide Hepatotoxicity in Rats Int. J. Bioeng. Life Sci. 2014 8 730 733
59. Siddiqi A. Hasan S.K. Nafees S. Rashid S. Saidullah B. Sultana S. Chemopreventive efficacy of hesperidin against chemically induced nephrotoxicity and renal carcinogenesis via amelioration of oxidative stress and modulation of multiple molecular pathways Exp. Mol. Pathol. 2015 99 641 653 10.1016/j.yexmp.2015.11.012 26551080
60. Filobbos S. Amin N. Yacoub M. Abd El_Hakim K.R. Possible Protective Effect of Melatonin on Cisplatin-Induced Testicular Toxicity in Adult Albino Rats. A Histological and Immunohistochemical Study Egypt. J. Histol. 2020 43 891 901 10.21608/ejh.2020.21561.1221
61. Zickri M.B. Moustafa M.H. Fasseh A.E. Kamar S.S. Antioxidant and antiapoptotic paracrine effects of mesenchymal stem cells on spermatogenic arrest in oligospermia rat model Ann. Anat. Anat. Anz. Off. Organ Anat. Ges. 2021 237 151750 10.1016/j.aanat.2021.151750 33940119
62. Wang Y. Bai L. Zhang J. Li H. Yang W. Li M. Lepidium draba L. leaves extract ameliorated cyclophosphamide-induced testicular toxicity by modulation of ROS-dependent Keap1/Nrf2/HO1, Bax/Bcl2/p53/caspase-3, and inflammatory signaling pathways J. Food Biochem. 2021 45 e13987 10.1111/jfbc.13987 34730245
63. Bashandy S.A.E. Ebaid H. Al-Tamimi J. Ahmed-Farid O.A. Omara E.A. Alhazza I.M. Melatonin Alleviated Potassium Dichromate-Induced Oxidative Stress and Reprotoxicity in Male Rats BioMed. Res. Int. 2021 2021 3565360 10.1155/2021/3565360 34222468 PMC8221856
64. Li L. Chen B. An T. Zhang H. Xia B. Li R. Zhu R. Tian Y. Wang L. Zhao D. BaZiBuShen alleviates altered testicular morphology and spermatogenesis and modulates Sirt6/P53 and Sirt6/NF-κB pathways in aging mice induced by D-galactose and NaNO(2) J. Ethnopharmacol. 2021 271 113810 10.1016/j.jep.2021.113810 33508368
65. Moradi M. Goodarzi N. Faramarzi A. Cheraghi H. Hashemian A.H. Jalili C. Melatonin protects rats testes against bleomycin, etoposide, and cisplatin-induced toxicity via mitigating nitro-oxidative stress and apoptosis Biomed. Pharmacother. = Biomed. Pharmacother. 2021 138 111481 10.1016/j.biopha.2021.111481 33752059
66. Li S. Che S. Chen S. Ruan Z. Zhang L. Hesperidin partly ameliorates the decabromodiphenyl ether-induced reproductive toxicity in pubertal mice Environ. Sci. Pollut. Res. Int. 2022 29 90391 90403 10.1007/s11356-022-20944-1 35871201
67. Kaya K. Ciftci O. Cetin A. Doğan H. Başak N. Hesperidin protects testicular and spermatological damages induced by cisplatin in rats Andrologia 2015 47 793 800 10.1111/and.12332 25220503
68. Celik E. Oguzturk H. Sahin N. Turtay M.G. Oguz F. Ciftci O. Protective effects of hesperidin in experimental testicular ischemia/reperfusion injury in rats Arch. Med. Sci. 2016 12 928 934 10.5114/aoms.2015.47697 27695481 PMC5016569
69. Elwan W.M. Kassab A.A. The Potential Protective Role of Hesperidin Against Capecitabine-Induced Corneal Toxicity in Adult Male Albino Rat. Light and Electron Microscopic Study Egypt. J. Histol. 2017 40 201 215 10.21608/EJH.2017.4077
70. Turk E. Kandemir F.M. Yildirim S. Caglayan C. Kucukler S. Kuzu M. Protective Effect of Hesperidin on Sodium Arsenite-Induced Nephrotoxicity and Hepatotoxicity in Rats Biol. Trace Elem. Res. 2019 189 95 108 10.1007/s12011-018-1443-6 30066062
71. El-Diasty H.H. El-Sayyad H. Refaat S. El-Ghaweet H.A. Efficacy of Quercetin-Sensitized Cisplatin against N-Nitroso-NMethylurea Induced Testicular Carcinogenesis in Wistar Rats Asian Pac. J. Cancer Prev. 2021 22 75 84 10.31557/APJCP.2021.22.1.75 33507682 PMC8184190
72. Adana M.Y. Imam A. Bello A.A. Sunmonu O.E. Alege E.P. Onigbolabi O.G. Ajao M.S. Oral thymoquinone modulates cyclophosphamide-induced testicular toxicity in adolescent Wistar rats Andrologia 2022 54 e14368 10.1111/and.14368 34997774
73. Ijaz M. Tahir A. Samad A. Ashraf A. Ameen M. Imran M. Yousaf S. Sarwar N. Casticin Alleviates Testicular and Spermatological Damage Induced by Cisplatin in Rats Pak. Vet. J. 2020 40 234 238 10.29261/pakvetj/2019.127
74. Delgarm N. Morovati-Sharifabad M. Salehi E. Afkhami-Ardakani M. Heydarnejad M.S. Exploring the main effects of phoenix dactylifera on destructive changes caused by cyclophosphamide in male reproductive system in mice Vet. Res. Forum Int. Q. J. 2022 13 249 255 10.30466/vrf.2022.540579.3250 PMC9340291 35919849
75. Bakhtiary Z. Shahrooz R. Ahmadi A. Soltanalinejad F. Ethyl Pyruvate Ameliorates The Damage Induced by Cyclophosphamide on Adult Mice Testes Int. J. Fertil. Steril. 2016 10 79 86 10.22074/ijfs.2016.4772 27123204 PMC4845533
76. Samie A. Sedaghat R. Baluchnejadmojarad T. Roghani M. Hesperetin, a citrus flavonoid, attenuates testicular damage in diabetic rats via inhibition of oxidative stress, inflammation, and apoptosis Life Sci. 2018 210 132 139 10.1016/j.lfs.2018.08.074 30179627
77. Alanbaki A. Al-Mayali H. Al-Mayali H. Ameliorative effect of Quercetin and Hesperidin on Antioxidant and Histological Changes in the Testis of Etoposide-Induced Adult Male Rats Res. J. Pharm. Technol. 2018 11 564 10.5958/0974-360X.2018.00105.1
78. Jahan S. Iftikhar N. Ullah H. Rukh G. Hussain I. Alleviative effect of quercetin on rat testis against arsenic: A histological and biochemical study Syst. Biol. Reprod. Med. 2015 61 89 95 10.3109/19396368.2014.998350 25539033
79. Aboelwafa H.R. Ramadan R.A. El-Kott A.F. Abdelhamid F.M. The protective effect of melatonin supplementation against taxol-induced testicular cytotoxicity in adult rats Braz. J. Med. Biol. Res. Rev. Bras. Pesqui. Med. Biol. 2022 55 e11614 10.1590/1414-431x2021e11614 PMC8851920 35137851
80. Arisha A.H. Moustafa A. Potential inhibitory effect of swimming exercise on the Kisspeptin–GnRH signaling pathway in male rats Theriogenology 2019 133 87 96 10.1016/j.theriogenology.2019.04.035 31075715
81. Osawe S.O. Farombi E.O. Quercetin and rutin ameliorates sulphasalazine-induced spermiotoxicity, alterations in reproductive hormones and steroidogenic enzyme imbalance in rats Andrologia 2018 50 e12981 10.1111/and.12981 29512831
82. Sharma P. Aslam Khan I. Singh R. Curcumin and Quercetin Ameliorated Cypermethrin and Deltamethrin-Induced Reproductive System Impairment in Male Wistar Rats by Upregulating The Activity of Pituitary-Gonadal Hormones and Steroidogenic Enzymes Int. J. Fertil. Steril. 2018 12 72 80 10.22074/ijfs.2018.5160 29334211 PMC5767937
83. Nayak G. Rao A. Mullick P. Mutalik S. Kalthur S.G. Adiga S.K. Kalthur G. Ethanolic extract of Moringa oleifera leaves alleviate cyclophosphamide-induced testicular toxicity by improving endocrine function and modulating cell specific gene expression in mouse testis J. Ethnopharmacol. 2020 259 112922 10.1016/j.jep.2020.112922 32422360
84. Nna V.U. Ujah G.A. Suleiman J.B. Mohamed M. Nwokocha C. Akpan T.J. Ekuma H.C. Fubara V.V. Kekung-Asu C.B. Osim E.E. Tert-butylhydroquinone preserve testicular steroidogenesis and spermatogenesis in cisplatin-intoxicated rats by targeting oxidative stress, inflammation and apoptosis Toxicology 2020 441 152528 10.1016/j.tox.2020.152528 32565124
85. Adelakun S.A. Akintunde O.W. Jeje S.O. Alao O.A. Ameliorating and protective potential of 1-isothiocyanato-4-methyl sulfonyl butane on cisplatin induced oligozoospermia and testicular dysfunction via redox-inflammatory pathway: Histomorphometric and immunohistochemical evaluation using proliferating cell nuclear antigen Phytomedicine Plus 2022 2 100268 10.1016/j.phyplu.2022.100268
86. Noshy P.A. Khalaf A.A.A. Ibrahim M.A. Mekkawy A.M. Abdelrahman R.E. Farghali A. Tammam A.A. Zaki A.R. Alterations in reproductive parameters and steroid biosynthesis induced by nickel oxide nanoparticles in male rats: The ameliorative effect of hesperidin Toxicology 2022 473 153208 10.1016/j.tox.2022.153208 35569531
87. Can S. Çetik Yıldız S. Keskin C. Şahintürk V. Cengiz M. Appak Başköy S. Ayhanci A. Akıncı G. Investigation into the protective effects of Hypericum Triquetrifolium Turra seed against cyclophosphamide-induced testicular injury in Sprague Dawley rats Drug Chem. Toxicol. 2022 45 1679 1686 10.1080/01480545.2020.1856130 33280440
88. Ye H. Ng H.W. Sakkiah S. Ge W. Perkins R. Tong W. Hong H. Pathway Analysis Revealed Potential Diverse Health Impacts of Flavonoids that Bind Estrogen Receptors Int. J. Environ. Res. Public Health 2016 13 373 10.3390/ijerph13040373 27023590 PMC4847035
89. Huang Z. Fang F. Wang J. Wong C.-W. Structural activity relationship of flavonoids with estrogen-related receptor gamma FEBS Lett. 2010 584 22 26 10.1016/j.febslet.2009.11.026 19914244
90. Cannarella R. Calogero A.E. Condorelli R.A. Greco E.A. Aversa A. La Vignera S. Is there a role for glucagon-like peptide-1 receptor agonists in the treatment of male infertility? Andrology 2021 9 1499 1503 10.1111/andr.13015 33818920 PMC8596505
91. Starovlah I.M. Radovic Pletikosic S.M. Kostic T.S. Andric S.A. Reduced spermatozoa functionality during stress is the consequence of adrenergic-mediated disturbance of mitochondrial dynamics markers Sci. Rep. 2020 10 16813 10.1038/s41598-020-73630-y 33033347 PMC7544694
92. Abdullah D.M. Alsemeh A.E. Khamis T. Semaglutide early intervention attenuated testicular dysfunction by targeting the GLP-1-PPAR-α-Kisspeptin-Steroidogenesis signaling pathway in a testicular ischemia-reperfusion rat model Peptides 2022 149 170711 10.1016/j.peptides.2021.170711 34920048
93. Almeer R.S. Abdel Moneim A.E. Evaluation of the Protective Effect of Olive Leaf Extract on Cisplatin-Induced Testicular Damage in Rats Oxid Med. Cell Longev. 2018 2018 8487248 10.1155/2018/8487248 30013722 PMC6022323
94. Gur C. Kandemir O. Kandemir F.M. Investigation of the effects of hesperidin administration on abamectin-induced testicular toxicity in rats through oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis, autophagy, and JAK2/STAT3 pathways Environ. Toxicol. 2022 37 401 412 10.1002/tox.23406 34748272
95. Negm W.A. El-Kadem A.H. Hussein I.A. Alqahtani M.J. The Mechanistic Perspective of Bilobetin Protective Effects against Cisplatin-Induced Testicular Toxicity: Role of Nrf-2/Keap-1 Signaling, Inflammation, and Apoptosis Biomedicines 2022 10 1134 10.3390/biomedicines10051134 35625871 PMC9138600
96. Selvaraj P. Pugalendi K.V. Efficacy of hesperidin on plasma, heart and liver tissue lipids in rats subjected to isoproterenol-induced cardiotoxicity Exp. Toxicol. Pathol. Off. J. Ges. Fur Toxikol. Pathol. 2012 64 449 452 10.1016/j.etp.2010.10.012 21095107
97. Al-Shahat A. Hulail M.A.E. Soliman N.M.M. Khamis T. Fericean L.M. Arisha A.H. Moawad R.S. Melatonin Mitigates Cisplatin-Induced Ovarian Dysfunction via Altering Steroidogenesis, Inflammation, Apoptosis, Oxidative Stress, and PTEN/PI3K/Akt/mTOR/AMPK Signaling Pathway in Female Rats Pharmaceutics. 2022 14 2769 10.3390/pharmaceutics14122769 36559263 PMC9786155
98. Zirkin B.R. Chen H. Regulation of Leydig cell steroidogenic function during aging Biol. Reprod. 2000 63 977 981 10.1095/biolreprod63.4.977 10993816
99. Moustafa A. Effect of Light-Dark Cycle Misalignment on the Hypothalamic-Pituitary-Gonadal Axis, Testicular Oxidative Stress, and Expression of Clock Genes in Adult Male Rats Int. J. Endocrinol. 2020 2020 1426846 10.1155/2020/1426846 33204259 PMC7666629
100. Livak K.J. Schmittgen T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 −ΔΔCT Method Methods 2001 25 402 408 10.1006/meth.2001.1262 11846609
101. Bancroft J.D. Layton C. 12—Connective and other mesenchymal tissues with their stains Bancroft’s Theory and Practice of Histological Techniques 8th ed. Suvarna S.K. Layton C. Bancroft J.D. Elsevier Amsterdam, The Netherlands 2019 153 175
102. Johnston D.S. Russell L.D. Friel P.J. Griswold M.D. Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation Endocrinology 2001 142 2405 2408 10.1210/endo.142.6.8317 11356688
103. Sharpe R.M. Sperm counts and fertility in men: A rocky road ahead. Science & Society Series on Sex and Science EMBO Rep. 2012 13 398 403 10.1038/embor.2012.50 22491033 PMC3343360
104. Jensen E.C. Quantitative analysis of histological staining and fluorescence using ImageJ Anat. Rec. 2013 296 378 381 10.1002/ar.22641 23382140