Impact of Gervital Against Histopathological, Ultrastructural, and Biochemical Alterations Caused by Methotrexate or Azathioprine in Albino Rats Testis

Methotrexate (MTX) and azathioprine (AZA) are chemotherapeutic, immunosuppressive, cytotoxic drugs with reported adverse impacts such as oxidative damage to testis. This study aims to scrutinize the potential effect of grape seed extract (GSE; gervital) to prevent testicular damage propelled by MTX and AZA. Rats were separated into six groups: Group I, normal control group; Group II, GSE (150 mg/kg/day); Group III, MTX (8 mg/kg/week). Group IV, AZA (15 mg/kg/day). Group V, GSE (150 mg/kg/day) + MTX (8 mg/kg /week); Group VI, GSE (150 mg/kg/day) + AZA (15 mg/kg/day). All rats were sacriced, blood samples were obtained for testosterone analysis and testis was removed for histological, ultrastructural studies and oxidation measurements. A reduction in body and testes relative weight, along with a signicant decrease in testosterone was observed. Histopathological, ultrastructural alterations induced by MTX or AZA include scanty spermatozoa, sloughing, marked reduction of spermatogonia cells, and pyknosis of some nuclei. Signicant oxidative stress (OS) manifested by reduced glutathione (GSH) level, catalase (CAT) and superoxide dismutase (SOD) as well as increase in malondialdehyde (MDA) levels. GSE administration showed an ameliorative effect on testosterone, histopathological and ultrastructural changes. GSE treatment also suppressed the increases in MDA levels and the decrease in GSH level, CAT, and SOD activities. Conclusion, our ndings conrmed that GSE is an effective antioxidant that prevent and protect testis from the histopathological and ultrastructural damages induced by MTX and AZA. So, GSE is a promising candidate for future use to minimize and alleviate MTX and AZA risks consumed in the and the world's major fruit crop al. is a potent antioxidant compound because it can surmount superoxide radicals in living cells and the main component of it is proanthocyanidins. It conserves cells from dangerous diseases thus it is considered as anti-diabetic, anti-tumor, anti-microbial, anti-aging and anti-inammation (Kwatra 2020). This study aimed to scrutinize the potential role of GSE to prevent testicular damage propelled by MTX AZA. Also, to clarify the ultrastructural changes induced by azathioprine, since little is known about it in the previous studies. appeared with damage to the structure of germ cells. Moreover, Ateşşahin et al. (2006) manifested that damage to the structure of germ cells caused by OS. The current AZA-treated group revealed degenerated seminiferous tubules, marked decrease in the spermatogenic cells, pyknotic nuclei, and vacuolated cytoplasm. The cytoplasmic vacuolation may be due to Sertoli cell damage, where Sertoli cells control the spermatogenic process as explained by recent research by Abdelbaky et al. (2020). The present ultrastructural study of the MTX group revealed several extreme changes in spermatogonia and Sertoli cells. Richburg (2000), manifested that when Sertoli cell numbers diminish, the number of germinal cells declines intensively. The present study also revealed cytoplasmic vacuolations and marked decreasing of sperm number in the lumen of the seminiferous tubule. Reduced relative testicular weight along with damage to the sperm DNA and sperm count (Padmanabhan et al. 2009) and spermatozoa have been highly susceptible to LP resulted from ROS due to the high contact polyunsaturated fatty acids as manifested previously by Alvarez et al. (1987). The current ultrastructural study of the AZA group revealed a germ cell with pyknotic nuclei and vacuolated cytoplasm. Degenerated Sertoli cells and spermatogonia cells with many vacuoles. This results in agreement with Karawya and El-Nahas (2006) who demonstrated the decrease in the testis weight is indirectly indicative of the effect on spermatogenesis. The current study also revealed a marked decrease in the number of sperms in the lumen. Similar results were observed by Onanuga et al. (2014). The present light microscopic examination and ultrastructural study of testis treated with GSE plus MTX and testis treated with GSE plus AZA showed nearly normal structure in most seminiferous tubules, improvement of spermatogenic cells, Sertoli cell, primary spermatocyte


Introduction
Chemotherapy is the utilize of chemical agents to suppress or kill proliferating cancer cells by several mechanisms, including cell membrane AZA is a cytotoxic immunomodulatory drug that is commonly used to treat in ammatory bowel disease, autoimmune disorders, organ transplant rejection, and cancer ( Reggio et al. 2019;Bergasa 2022)). AZA leads to interstitial spaces to be poorly de ned, scanty spermatogenic cells and spermatozoa, disrupted, and shrunken seminiferous tubules (Shaikh et al. 2020b).
Antioxidants are considered the rst line of defense against free radical destruction and are perfect for keeping ideal health and well-being. They play an animated role against reactive oxygen species (ROS) in the body's defense system (Hasanuzzaman et al. 2020). Grape (Vitis vinifera) is one of the greatest frequently consumed fruits in the world and the world's major fruit crop (Raja et al. 2020).
Grape seed extract (GSE); gervical is a potent antioxidant compound because it can surmount superoxide radicals in living cells and the main component of it is proanthocyanidins. It conserves cells from dangerous diseases thus it is considered as anti-diabetic, anti-tumor, anti-microbial, anti-aging and anti-in ammation (Kwatra 2020). This study aimed to scrutinize the potential role of GSE to prevent testicular damage propelled by MTX or AZA. Also, to clarify the ultrastructural changes induced by azathioprine, since little is known about it in the previous studies.

Animals and experimental design
A total of thirty-six Wistar rats; 140-200 g, aged 6-8 weeks old, were collected from Nahda University Animal House Facility Center. All experimental rats were housed and maintained in a clean rodent room and well ventilated under standard conditions of temperature and humidity, room temperature (25 ± 5 ºC) with 12-h dark/light cycles and were received free access to a standard diet of pellets and tap water. They were saved under observation for 15 days before the experiment started to rule out any intercurrent infections. Animals received human care in compliance with the recommendations of the Committee for the control Purpose, Care animals follow the European Community Directive (86/609 / EEC Edition 8). This has already been approved Committee of Zoology, Beni-Suef University, Egypt. IACUC (permit number BSU/FS/2018/1).

Animal grouping
Rats were separated into six groups (n= 6). Group I, normal control group. Group II (GSE); animals were orally given GSE (150 mg/kg body weight /day) dissolved in distilled water according to previous research (Bagchi et al. 2001). Group III (MTX); animals received an oral dose of MTX (8 mg/kg body weight /week) dissolved in 5% tween 80 and saline once a week (Akinlolu et al. 2014). Group IV (AZA); animals received an oral daily dose of the solutions of AZA (15 mg/kg body weight/day) were kept in saline containing 5% Tween 80 acquiesced with previous research (Akinlolu et al. 2014) Group V (GSE +MTX); rats were treated with a dose of GSE then followed after 2 h by an oral dose of MTX. Group VI (GSE +AZA); animals were administered with a dose of GSE then followed after 2 h by an oral dose of AZA.

Sample collection and the biochemical estimation
Rats were sacri ced after 35 days of the experiment under light diethyl ether anesthesia. Each rat was examined weekly for bodyweight investigation. With the aid of a capillary tube, blood samples were taken from the retro-orbital venous plexus into serum tubes (Kumar et al. 2011).

Tissue homogenate preparation and oxidative stress-related markers assay
The testes were rapidly expelled from the surrounding tissues and weighed. Testis homogenate 25% in isotonic ice-cooled normal saline. The resulting homogenate was centrifuged for 15 mins at a speed of 10.000 rmp. At -20 ° C, the supernatant was preserved before analysis. The testicular homogenate was used for malondialdehyde (MDA) estimation according to Altintas et al. (2014). Testicular superoxide dismutase (SOD) and catalase (CAT) activities (Marklund & Marklund 1974); (Claiborne 1985) were measured respectively. Glutathione (GSH) was calculated by using the method of previous research (Sedlak & Lindsay 1968).

Histological examination
Testis tissues were cut into 0.5 cm 3 small pieces and then xed for 24 h in 10% neutral formalin buffer. Testis specimens were cleaned to remove the residual xative and then dehydrated for 45 minutes each in ascending grades of ethanol, then for 30 minutes each in two levels of absolute ethyl alcohol. This was followed by clearance of 30 minutes each in two xylene changes. The tissues were then soaked for three hours at 60°C with paraplast plus (three changes) and then embedded in paraplast plus. For histopathological tests, sections 4 to 5 µm thick were equipped with a microtome and stained with Hematoxylin and eosin (H&E) (Bancroft & Gamble 2008).

Ultrastructural examination
Testis specimens were cut into small pieces measuring about 1 mm 3 and immediately xed for 18-24 hours in fresh 3% glutaraldehydeformaldehyde at 4 o C. The specimens were then washed in a phosphate buffer (pH 7.4) and then set for one hour at 4 o C in isotonic 1% osmium tetroxide. (Bain & Mercer 1966). A Series of alcohol dehydration was done. The specimens were then transmitted through a propylene oxide solution 2 times in 10 minutes each. Embedding of the specimens in Epon epoxy resin started by in ltrating the specimens in the propylene oxideresin mixture overnight. The specimens were transferred to fresh-resin capsules for polymerization to obtain hard blocks. Sections of Semithin were cut by ultracut Reichert-Jung ultramicrotome from these blocks with the aid of glass knives, stained with toluidine blue stain. According to prior research. (Bozzola & Russell 1999) ultrathin sections were then prepared and stained with uranyl acetate and lead citrate, and examined with a Joel CX 100 transmission electron microscope operating at an accelerating voltage of 60 KV.

Statistical analysis
The Statistical Package for the Social Sciences (SPSS, version 20.0 for WINDOWS; SPSS, Chicago, IL). The results were elucidated as the mean ± standard error and all statistical comparisons were calculated using one-way ANOVA test (Rao et al. 1985) followed by Duncan's multiple range test analysis values. The signi cant difference is counted at P-value< 0.05.

Body weight
MTX and AZA therapy affected the percentage of change between the two body weights, where the rats suffered from a marked decrease (P< 0.05) as compared to the control and GSE groups. While GSE showed maintenance of body weight in MTX plus GSE group (Fig. 1).

Testes relative weight
MTX and AZA therapy affected relative testis weight, where the rats suffered from a marked decrease (P< 0.05) in testis relative weight when compared to the control and GSE groups. While co-administration of MTX with GSE or AZA with GSE conserves the decrease in testis relative weight (Fig. 2).

Testosterone determination
A signi cant decrease in testosterone (P< 0.05) in MTX and AZA groups in comparison to the control and GSE groups. GSE treatment produced a potential amelioration of testosterone for both MTX and AZA treated groups (Fig. 3).

Testis antioxidant parameters and oxidative stress
Lipid peroxidation (LP), articulated as MDA concentration was measured as a biomarker of testis OS state. It is demonstrated a signi cant increase in MTX and AZA treated groups when compared to control and GSE treated ones. GSE treatment agent produced a potential improvement of the LP level for both MTX and AZA treated groups. Concerning the enzymatic and non-enzymatic antioxidant defense system, CAT, SOD, and GSH all revealed a noticeable reduction in MTX and AZA groups (P< 0.05). On the other hand, GSE improved the activities of these antioxidant enzymes and increased GSH levels in the treated groups (Table 1). Table 1 The potential preventive effect of GSE against MTX and AZA induced changes in MDA level, CAT, SOD activity and GSH concentration in testis tissue of all experimental groups.

Histopathological changes
Examination of testicular tissue of both control and GSE-treated animals revealed normal seminiferous tubules with active spermatogenesis.
Spermatogonia and triangular Sertoli cell resting upon the basement membrane. Leydig cell and cluster of spermatozoa were seen in lumen.
Primary spermatocytes were recognized by their large nuclei containing coarse clumps of chromatin, and spermatids appeared with rounded nuclei in control ( Fig. 4a and b) and GSE ( Fig. 4c and d).
Animals treated with MTX showed severe degenerated and variable-sized seminiferous tubules, where many tubules appeared with a marked decrease in the spermatogenic cells and few or no sperms. Detachment of spermatogenic cells from the basal lamina and vacuolated cytoplasm was observed. There was sloughing of spermatogenic cells into the lumen of seminiferous tubules (Fig. 4e).
Animals treated with AZA revealed degenerated seminiferous tubules with ruptured basement membrane were observed. Notice the detachment of spermatogenic cells from the basal lamina and vacuolated cytoplasm. The reduction of spermatogenic cells and pyknosis of some nuclei were also seen (Fig. 4f).
Examination of testes of animals treated with GSE plus MTX revealed approximate recovery of seminiferous tubules amelioration of spermatogenic cells, and spermatozoa except few vacuoles were observed. Normal Leydig cells were seen in the interstitial tissue (Fig. 4g).
Examination of testes of animals treated with GSE and AZA showed improvement of spermatogenesis, nearly normal structure in most seminiferous tubules. Compact spermatogenic layers with few degenerative germ cells and normal Leydig cells were seen (Fig. 4h).

Ultrastructural changes
The electron microscopic examination of the control testis revealed the spermatogonia rested on the basement membrane, myoid cell, the Sertoli cell had a triangular nucleus, primary spermatocyte with mitochondria, and large spherical nucleus (Fig. 5a). There was a Rounded Spermatid with spherical nuclei, acrosomal cap, and peripherally located mitochondria were observed (Fig. 5b). In addition to lumen contains normal spermatozoan at the midpiece and tail region (Fig. 5c). Normal interstitial tissue containing Leydig cell with a large nucleus, thin rim of chromatin, prominent nucleolus, lipid droplet, and capillaries (Fig. 5d).
The ultrastructure observation of testes of animals administered with MTX revealed many vacuoles and vacuolated mitochondria in spermatogonia, Sertoli cell, and primary spermatocyte. Presence of some lysosome in the Sertoli cells and spermatogonia (Fig. 6a). Distorted spermatid with marked cytoplasmic vacuolation and rari ed cytoplasm surrounded by a large lytiareashe presence of some lysosome and some degenerated mitochondria was observed (Fig. 6b). A marked decrease in the number of sperms in the lumen of the seminiferous tubule (Fig. 6c).
Abnormal interstitial tissue with degenerated Leydig cell with an irregular nucleus having a thick rim of heterochromatin, few lipids drop and dilated smooth endoplasmic reticulum were also seen (Fig. 6d).
The ultrastructure observation of testes of animals administered with AZA showed many vacuoles in both Sertoli cells and spermatogonia cells and an overall decrease in cytoplasmic ground substance (Fig. 7a). Thick ruptured irregular basement membrane, vacuolated primary spermatocyte, and severe degenerated Sertoli cells with many vacuoles and some lysosomes were detected (Fig. 7b). There was distorted and vacuolated early spermatid. Notice the dissolution of some cells (Fig. 7c). Degenerated vacuolated spermatid and dissolution of some cells were observed (Fig. 7d). In addition to degenerated Leydig cell. Notice the irregular nuclear envelope with dark clumps of heterochromatin adjacent to the nuclear membrane and notice presence of few lysosomes (Fig. 7e).
The ultrastructure observation of testes of animals administered with GSE and MTX showed amelioration in the structure of spermatogonium, Sertoli cell and primary spermatocyte except few degenerated mitochondrial (Fig. 8a). Normal spermatid appeared with a rounded nucleus and peripherally located mitochondria except a few cytoplasmic vacuolations and a few lysosomes (Fig. 8b). Lumen with marked recovery in transverse sections of normal sperms at mid piece and tail region (Fig. 8c). Leydig cells with a large nucleus, smooth endoplasmic reticulum, lipid droplets, and blood capillaries were seen (Fig. 8d).
The ultrastructure observation of testes of animals administered with GSE and AZA revealed a slightly normal basement membrane, improvement in the structure of cells except vacuolation in Sertoli cell, spermatogonia, primary spermatocyte, and spermatid which with a rounded nucleus and normal acrosomal cap (Fig. 9a). In addition, around spermatid appeared with an acrosomal cap, peripherally located mitochondria, and few cytoplasmic vacuolations (Fig. 9b). Round spermatid and a moderate number of cross-sections at the midpiece and tail region of sperms were seen in the lumen (Fig. 9c). Interstitial tissue and Leydig cell with a normal nucleus and a moderate number of lipid droplets were observed (Fig.   9d).

Conclusion
The present study determined that GSE had preventive effects against MTX or AZA Prompted testicular damage that was con rmed through histological, ultrastructure and biochemical studies. The preventive effect of GSE may be attributed to its antioxidant potential against free radicals. Our study revealed marked amelioration after treatment with GSE on toxicity induced by MTX and AZA, but there were slight improvements in MTX-treated group compared to AZA-treated group.

Declarations
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