Research Article

Histopathological and PCNA-Based Immunohistochemical Assessment of Pumpkin and Grape Seed Extracts in Protecting Male Rat Accessory Glands Under Chemotherapeutic Stress

Ibrahim Elmaghraby1, Zeinab Said2, Marwa Darweish1, Doaa Galal El-Sahra3 and Ahmed Ibrahim El-Nemr4*

1Department of Pathology, Faculty of Veterinary Medicine, Benha University, Toukh 13736, Egypt; 2Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt; 3Medical Surgical Nursing Department, Faculty of Nursing, Modern University for Technology and Information, Cairo, Egypt; 4Medical Laboratory Technology Department, College of Health and Medical Techniques, Almaaqal University, Basra 61003, Iraq.

Received | December 27, 2024; Accepted | February 04, 2025; Published | February 15, 2025

*Correspondence | Ahmed Ibrahim El-Nemr, Medical Laboratory Technology Department, College of Health and Medical Techniques, Almaaqal University, Basra 61003, Iraq; Email: [email protected]

Citation | Elmaghraby, I., Z. Said, M. Darweish, D.G. El-Sahra and A.I. El-Nemr. 2025. Histopathological and PCNA-based immunohistochemical assessment of pumpkin and grape seed extracts in protecting male rat accessory glands under chemotherapeutic stress. Advanced Analytical Pathology, 1: 12-21.

DOI | https://dx.doi.org/10.17582/journal.ppa/2025/1.12.21

Keywords | Cyclophosphamide, Pumpkin seed extract, Grape seed extract, Epididymis, Prostate, Seminal vesicle

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Male fertility relies on a complex interplay of reproductive organs, with the epididymis and accessory glands (prostate and seminal vesicles) serving as crucial components in this intricate system. The epididymis plays a dual role by not only storing sperm but also providing essential protection through the secretion of antioxidant enzymes that neutralize reactive oxygen species O’Flaherty (2019). These specialized glands contribute significantly to seminal plasma composition, producing vital secretions that ensure sperm mobility, provide nourishment, facilitate capacitation, and promote survival (Verze et al., 2016; Noda and Ikawa, 2019; Wang et al., 2020).

In the context of cancer treatment, chemotherapy poses significant challenges to male reproductive health, often resulting in temporary or permanent fertility impairment. Cyclophosphamide (CP), a widely prescribed alkylating agent in the treatment of neoplastic and autoimmune diseases, exemplifies this therapeutic challenge (Ghafouri-Fard et al., 2021; Ayza et al., 2022). Despite its therapeutic efficacy, CP’s clinical application is constrained by its pronounced reproductive dysfunction and cytotoxic effects Pavin et al. (2018).

The reproductive toxicity of CP manifests through multiple pathways, including disrupted gonadotropin secretion, hormonal dysregulation, severe oxidative stress in tissues, extensive cellular damage, ATP depletion, and genotoxic effects Ghobadi et al. (2017). Mechanistically, CP functions as a prodrug, undergoing hepatic metabolism to generate two active compounds: Phosphoramide mustard, responsible for therapeutic effects, and acrolein, which induces toxicity through apoptosis and necrosis in normal tissues Gu et al. (2017). To mitigate these adverse effects while potentially enabling higher therapeutic doses, antioxidant supplementation has emerged as a promising strategy Cetik-Yildiz et al. (2015).

Natural compounds with antioxidant properties have gained attention as potential protective agents. Grape (Vitis vinifera L.), one of the world’s most cultivated fruit crops, offers exceptional nutritional and therapeutic benefits due to its rich antioxidant profile. The seed extract has a strong antioxidant activity since it contains 60–70% polyphenolics Moosavi et al. (2015). Grape seed extract (GSE) is characterized by high concentrations of vitamins C and E and bioflavonoids, providing robust protection against oxidative injury, inflammatory mediators, and carcinogenic effects Alkhedaide et al. (2016). Beyond its antioxidant properties, GSE demonstrates diverse pharmacological activities, including antibacterial, antiviral, anti-inflammatory, anti-allergic, and vasodilatory effects, while also showing promise in protecting reproductive function Martins et al. (2021).

Similarly, pumpkin (Cucurbita pepo) has emerged as a significant medicinal plant with considerable nutritional value Shaban and Sahu (2017). Pumpkin seed extract (PSE) presents a rich nutritional profile, containing essential trace elements like zinc, vital vitamins (carotenoids, tocopherol), proteins, phytosterols, and polyunsaturated fatty acids Stevenson et al. (2007). Notably, PSE’s high oleic acid content, a monounsaturated fatty acid, helps protect reproductive organs against lipid peroxidation Shaban and Sahu (2017). The therapeutic applications of pumpkin extend to antineoplastic, antioxidant, antibacterial, anti-diabetic, and anti-obesity properties Wadi et al. (2021). Research has demonstrated that PSE’s antioxidative properties can enhance fertility and prevent reproductive dysfunction (Aghaei et al., 2014; Mohamadi et al., 2014).

Given the established antioxidant and protective properties of both GSE and PSE, coupled with the significant reproductive toxicity associated with cyclophosphamide treatment, this study aims to investigate the potential protective effects of these natural extracts against CP-induced gonadotoxicity. This research seeks to provide insights into possible therapeutic strategies for preserving male reproductive function during chemotherapy.

Materials and Methods

Experimental animals

Adult male Albino rats (n=36, weight: 270 ± 20 g) were obtained from the Animal House Facility, Faculty of Veterinary Medicine, Zagazig University, Egypt. Animals were maintained under controlled laboratory conditions (temperature: 25±5°C; relative humidity: 55 ± 5%; 12-hour light/dark cycle) with standard laboratory diet and ad libitum access to water. Prior to experimentation, animals underwent a one-week acclimatization period. All experimental procedures were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) Faculty of Nursing, Modern University for Technology, and Information (MTI), Egypt with a formal approval number (FAN/140/2024).

Pharmaceuticals and extract preparation

Cyclophosphamide: Cyclophosphamide (Endoxan®, Baxter Oncology GmbH, Germany) was obtained in its commercial form and prepared according to manufacturer’s specifications.

Grape seed extract (GSE) preparation: Grape seeds (Vitis vinifera L.) were manually isolated, shade-dried (25–30°C, 7 days), and pulverized to fine powder using an electric grinder. The powder underwent maceration in 70% ethanol (1:4 w/v ratio) for 72 hours at room temperature (25 ± 2°C) with intermittent agitation. The mixture was filtered through Whatman No. 1 filter paper, and the filtrate was evaporated to dryness under reduced pressure using a rotary evaporator (temperature: 40°C) to obtain GSE powder modified from Badavi et al. (2013).

Pumpkin seed extract (PSE) preparation: Cucurbita pepo seeds were manually collected, washed with distilled water, and air-dried. The dried seeds were finely ground, and the powder (100 g) was extracted with 80% ethanol (1:5 w/v) under continuous agitation. The mixture underwent vacuum filtration through Whatman No. 2 filter paper, followed by concentration using a rotary evaporator (40°C, under reduced pressure). The resulting PSE was reconstituted in sterile distilled water prior to administration modified from Xanthopoulou et al. (2009).

Experimental design

Thirty-six rats were randomly allocated into six experimental groups (n=6/group):

• Group I (Control): Received physiological saline (0.5 mL/kg, i.p.) daily from day 8-14
• Group II (CP): Administered cyclophosphamide (20 mg/kg, i.p.)
• Group III (PSE): Received pumpkin seed extract (600 mg/kg, p.o.)
• Group IV (GSE): Administered grape seed extract (300 mg/kg, p.o.)
• Group V (CP+PSE): Received CP (20 mg/kg, i.p.) + PSE (600 mg/kg, p.o.)
• Group VI (CP+GSE): Administered CP (20 mg/kg, i.p.) + GSE (300 mg/kg, p.o.)

GSE and PSE were administered orally from the first day of the experiment for 14 consecutive days. CP administration was initiated on day 8 and continued for 7 consecutive days. Dosages were selected based on previous studies (Sabik and Abd El‐Rahman, 2009; Aghaei et al., 2014; Hussien et al., 2013).

Histopathological and immunohistochemical analyses

Tissue processing and histopathology: The epididymis, seminal vesicle, and prostate were carefully excised and fixed in Bouin’s solution (24 hours). Tissues were processed according to standard histological protocols and embedded in paraffin. Serial sections (5 μm thickness) were prepared and stained with: a) Hematoxylin and Eosin (H&E) for routine histopathological examination b) Masson’s trichrome for evaluation of extracellular matrix deposition Bancroft and Layton (2019).

Immunohistochemical analysis: PCNA immunostaining was performed using mouse monoclonal anti-PCNA antibody (Clone PC 10, DAKO A/S, Denmark; 1:200 dilution in Tris-buffered saline). Deparaffinized sections underwent overnight incubation with primary antibody (4°C), followed by visualization using an avidin-biotin-peroxidase detection system. 3,3’-Diaminobenzidine (DAB) was used as chromogen, and sections were counterstained with hematoxylin. All immunohistochemical procedures were performed simultaneously under standardized conditions D’Andrea et al. (2008).

Microscopic analysis: Histopathological and immunohistochemical evaluations were performed using a Nikon E800 Eclipse light microscope equipped with a digital imaging system.

Statistical analysis

Data analysis was performed using GraphPad Prism software (version 9, La Jolla, CA, USA). Results are expressed as mean ± standard error of the mean (SEM). Intergroup comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. Statistical significance was set at P < 0.05.

Results and Discussion

Effect of PSE and GSE on epididymal, prostatic and seminal vesicle weight

Compared with control group, CP group showed a significant (p < 0.001) reduction in epididymal and prostatic weights as well as seminal vesicle weight (p < 0.0001). Meanwhile, rats pretreated with GSE or PSE showed a significant improvement (p < 0.05) in epididymal and (p < 0.01) in prostate and seminal vesicle when compared to the CP group.

 

Histopathological assessment of epididymal tissue

Histological examination of the epididymis showed circular and oblong epididymal tubules that are lined with stereociliated columnar epithelium, properly surrounded by fibromuscular connective tissue. Abundant healthy spermatozoa were visible in the lumen of the epididymal tubules in the control group (Figure 2A). Similar histological structure was observed in PSE and GSE groups. Administration of CP resulted in severe disruption in the epididymal tissue evidenced by congestion accompanied by edema and hemorrhage in between the epididymal tubules. Vacuolation, disorganization, and desquamation of the epididymal epithelial cells with necrotic sperm in the tubular lumen was also observed. Some epididymal tubules suffered from hyperplasia as well as severe necrosis of their lining epithelial cells with thickening in the connective tissue layers in between epididymal tubules that confirmed by Masson trichrome stain (Figure 2B-H). While rats given CP and pretreated with GSE restored the structural architecture of epididymal tubules with mild oedema in between these tubules, and some necrotic sperm inside the lumen (Figure 2I-J). The pretreated rats with PSE showed a greater improvement with mild vacuolation in the epithelial lining of epididymal tubules with mild oedema between the affected tubules (Figure 2K-L).

Histopathological assessment of prostatic tissue

Histopathological examination of the prostate gland in the control group showed closely packed acini of different sizes. The acini were lined with tall columnar cells with oval nuclei and occasional papillary infoldings.

 

The lumina was filled with homogenous acidophilic secretion. Narrow inter-acinar spaces occupied by fibro-muscular stroma separated the acini (Figure 3A). Similar histological structure in GSE, and PSE groups. In the CP group, Significant histological changes in the prostate tissue’s acini and stroma. The affected acini displayed destructed epithelial cells, which had small pyknotic nuclei and detached into the lumen (Figure 3B). Some prostatic acini revealed marked hyperplasia of their lining epithelium with papillary projection into the acinar lumen. These arborization folds have darkly stained nuclei with vacuolated cytoplasm (Figure 3C). Occasionally, some acini lost their wall integrity and appeared atrophied with a reduction in their size, remarkable thinning of the lining epithelium, and increased stromal prominence (Figure 3D). Concerning the fibromuscular stroma separating the acini was thicker than the control group and exhibited stromal hyperplasia with hyaline plaques (Figure 3E). Moreover, some stromal areas revealed broad inter-acinar spaces, noticeable hemorrhage, faint proteinaceous edematous fluid, and infiltration of inflammatory cells. Most acini had scanted or absent prostatic secretions, some containing inflammatory cells in their lumina (Figure 3F). While rats given CP and pretreated with GSE restored the normal appearance of prostatic folding and secretion with some stromal widening (Figure 3G). Furthermore, a group given CP and pretreated with PSE revealed a great improvement in the prostate structure with abundant eosinophilic secretion in the lumen (Figure 3H). Masson’s trichrome staining was used to assess the fibromuscular stroma that separated the acini, which was shown by collagen deposition. The prostatic interstitial tissue of the CP group showed a significant accumulation of collagen in comparison to the normal control group. In contrast to the CP group, the pretreatment group’s either PSE or GSE prostatic tissue had substantially less collagen deposition (Figure 3I-L).

 

Histopathological assessment of seminal vesicles

The histological structure of the seminal vehicles was found to be normal in the control group. The gland was divided into lobes of varying shapes, sizes, and numbers by well-developed fibromuscular capsules and trabeculae. The parenchyma had a honeycomb-like appearance with visible anastomosing mucosal folds. The mucosa is formed of pseudostratified columnar epithelium and lamina propria. The columnar epithelial cells had acidophilic cytoplasm, and basal basophilic nuclei. Additionally, deeply stained luminal acidophilic secretion was observed (Figure 4A). Similar histological structure was detected in GSE and PSE treated groups. The CP-treated group revealed a significant atrophy of the seminal vesicles. The glands exhibited a reduction in glandular components and lining epithelial cells with the formation of small papillary folds, along with prominent fibromuscular stroma (Figure 4B). Furthermore, some alveoli exhibited a reduction in mucosal fold height, and papillary folds appeared flattened with diminished epithelial cells size (Figure 4C). In other examined sections of seminal vesicle, the lining epithelium had necrosis, with detached epithelial cells in the lumina. These lumina showed little acidophilic secretion. Additionally, there was a prominent fibromuscular stroma and monocellular inflammatory cell infiltrations (Figure 4D). The variant areas of fibromuscular stromal prominence expressed positive color by Masson trichrome stain (Figure 4E). Also noted widespread congestion and edema between the degenerated lobules (Figure 4F). Obviously, these alterations were seen in that group

 

were improved in the pretreated groups with GSE or PSE. These pretreated groups showed a restoration of the seminal vesicle’s tissues to their normal histological features, including normal epithelium, mucosal folds, secretion, and the reversal of most of the necrotic changes (Figure 4G, H).

Immunohistochemical assessment of epididymis, prostate, and seminal vesicle

Immunohistochemical examination of epididymis, prostate and seminal vesicle revealed a significant reduction in the number of immunopositive cells observed in CP treated group in comparison with the control group. In contrast, the pretreated groups (CP+GSE, CP+PSE) showed a significant increase in the number of PCNA-positive cells compared to the CP group (Figure 5).

 

Chemotherapy is an effective cancer treatment. However, it is related to cytotoxic effects on normal cells and tissues, especially in the reproductive system. Chemotherapy causes changes in fertility, organ structure, sexual hormones, and function, as well as quality of life Haghi-Aminjan et al. (2017). The susceptibility of the genital organ and accessory glands to chemotherapeutic agents is related to their high content of polyunsaturated membrane lipids, low oxygen tensions, and abundant ROS-generating systems Agarwal et al. (2014). Cyclophosphamide is an effective anticancer and immunosuppressive agent, but it is severely limited by reproductive toxicity as documented in a variety of species Ghobadi et al. (2017). This study highlights the possible protective roles of GSE and PSE in male rat accessory glands exposed to chemotherapy.

In the present study, CP induced various histopathological disruptions in the examined epididymis, prostate, and seminal vesicle glands. This result was consistent with Iqubal et al. (2020). These damages induced by CP were reflected in the wights of the examined organs that significantly decreased when compared to the control group. This finding correlated with Das et al. (2002) who noticed that CP caused a decrease in the male accessory gland weights due to inhibition in testicular androgenesis secretion. In the present investigation, the epididymal tissue of CP-treated rats exhibits a reduction in the luminal spermatozoa with hyperplasia, vacuolation, and necrosis of the epithelial lining cells that sloughed into the lumen. There was thickening in the fibromuscular connective tissue layer of almost all the epididymal tubules. Such findings coincide with those of previous reports Aghaei et al. (2014). The prostatic acini were destructed and atrophied with flattened epithelial lining. Some acini displayed hyperplastic lining epithelium with papillary projection into the acinar lumen. This result was consistent with Abdel-Hafez et al. (2017). The seminal vesicle of CP-treat rats was atrophied with necrosed detached lining epithelium along with little acidophilic secretion in the lumen. Additionally, there was a prominent fibromuscular stroma infiltrated with inflammatory cells. Similar results were recorded by Iqubal et al. (2020). The prominent fibromuscular stroma could be a contributing factor to the development of glandular atrophy, where the epithelial cells of the seminal vesicles are replaced by muscular tissue Do Nascimento et al. (2020). This deleterious effect of CP is mediated by oxidative stress, and DNA damage of the cells Fusco et al. (2021). Oxidative stress is considered a main modulator of inflammation, myofibroblast differentiation and extracellular matrix production (Richter and Kietzmann, 2016; Thuan et al., 2018). That explained the observed interstitial inflammatory cell infiltration, congested capillaries, edema, abundant smooth muscles, and hyaline material deposition in the examined organs. However, the epithelial hyperplasia in this work represents either a form of cellular adaptation to stress, or unfortunately, it takes place at the expense of normal function (Kumar et al., 2017).

In the current work revealed positive expression of PCNA in all groups in this study except in CP treated group. This could be related to the necrotic and atrophic changes observed in the epithelium Oltra et al. (2014). On the other hand, a significant increase in PCNA-positive cells was noticed in the pretreated groups with GSE or PSE indicating cellular proliferation. Earlier studies confirmed immunohistochemical findings using different markers for cellular proliferation, specifically Ki-67 in the cisplatin-treated group, Ki-67 was expressed in a low number of epithelial cells in the affected glands Abou-Elghait et al. (2022).

In the current study, pretreatment with GSE significantly reduced epididymal, prostatic, and seminal vesicle histological injury caused by CP and restored the weights of these organs. These findings were in parallel with Hasseeb et al. (2013) who detected the ameliorating effect of the GSE especially in high levels against the induced lesions of the Acrylamide in the male reproductive organs of the rats. This could be attributed to the high antioxidant activity of GSE due to the presence of phenolic compounds such as catechin, gallic acid, epicatechin, and pro-anthocyanidins which present a higher antioxidant capacity than vitamin C or E Gupta et al. (2020). Furthermore, it possess anti-inflammatory, and anti-apoptotic properties Martins et al. (2021).

Pretreatment with PSE markedly reversed the epididymal histological alterations induced by CP with abundant healthy luminal spermatozoa. These findings were in collaboration with Aghaei et al. (2014). As well as the pretreatment with PSE negated the adverse effect of CP on the prostate gland and seminal vesicle histological disruptions with restoration of the weight of these organs. The ameliorative effects of PSE could be contributed to presence of trace minerals such as selenium and zinc, which are powerful antioxidants Dowidar et al. (2020). Prostate health and function are dependent on high levels of zinc, which are required for the production and secretion of citrate, a major component of prostatic fluid Costello and Franklin (2016).

Furthermore, pumpkin seeds contain high antioxidant vitamins like tocopherol and carotenoid that playing a protective role against toxic substances and free radicals Lestari and Meiyanto (2018). Pumpkin seeds have an abundance of Phytosterols, they are used in benign hypertrophic prostate disease in which they preventing the transformation of testosterone to the more efficient androgen dihydrotestosterone (DHT) Arora et al. (2023).

Conclusions and Recommendations

Based on histopathological and immunohistochemical findings obtained from this study, it could be concluded that CP caused marked disruption in the accessory male sexual glands and epididymis that may be minimized by the pretreatment of GSE and PSE. The ameliorative effect of these seed extracts was related to their antioxidant efficacy.

Acknowledgement

The authors express their sincere gratitude to the Faculty of Veterinary Medicine, Benha University, and the Faculty of Nursing, Modern University for Technology and Information (MTI), Egypt, for providing the necessary facilities and support for this research. Special thanks are extended to the laboratory staff for their technical assistance.

Novelty Statement

This study provides novel evidence of the protective effects of pumpkin seed extract (PSE) and grape seed extract (GSE) against cyclophosphamide-induced reproductive toxicity in male rats. The research highlights the histopathological and PCNA immunohistochemical findings, demonstrating that both extracts significantly restore accessory gland structure and function, suggesting their potential as therapeutic agents to mitigate chemotherapy-induced gonadotoxicity.

Author’s Contribution

Ibrahim Elmaghraby conceptualized the study, designed the methodology, and performed the histopathological analysis. Zeinab Said contributed to the experimental design, data interpretation, and manuscript writing. Marwa Darweish conducted the laboratory work, including immunohistochemistry and data acquisition. Doaa Galal El-Sahra performed the statistical analysis, literature review, and manuscript editing. Ahmed Ibrahim El-Nemr supervised the research, reviewed the final manuscript, and handled correspondence. All authors read and approved the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Abdel-Hafez, S.M.N., Rifaai, R.A. and Abdelzaher, W.Y., 2017. Possible protective effect of royal jelly against cyclophosphamide-induced prostatic damage in male albino rats; a biochemical, histological and immuno-histo-chemical study. Biomed. Pharmacoth., 90: 15-23. https://doi.org/10.1016/j.biopha.2017.03.020

Abou-Elghait, A.T., Elgamal, D.A., Abd El-Rady, N.M., Hosny, A., Abd El-Samie, E.Z.A.A. and Ali, F.E.M., 2022. Novel protective effect of diosmin against cisplatin-induced prostate and seminal vesicle damage: Role of oxidative stress and apoptosis. Tissue Cell, 79: 101961. https://doi.org/10.1016/j.tice.2022.101961

Agarwal, A., Virk, G., Ong, C. and Du-Plessis, S.S., 2014. Effect of oxidative stress on male reproduction. World J. Mens Health, 32(1): 1-17. https://doi.org/10.5534/wjmh.2014.32.1.1

Aghaei, S., Nikzad, H., Taghizadeh, M., Tameh, A.A., Taherian, A. and Moravveji, A., 2014. Protective effect of Pumpkin seed extract on sperm characteristics, biochemical parameters and epididymal histology in adult male rats treated with Cyclophosphamide. Andrologia, 46(8): 927-935. https://doi.org/10.1111/and.12175

Alkhedaide, A., Alshehri, Z.S., Sabry, A., Abdel-Ghaffar, T., Soliman, M.M. and Attia, H., 2016. Protective effect of grape seed extract against cadmium-induced testicular dysfunction. Mol. Med. Rep., 13(4): 3101-3109. https://doi.org/10.3892/mmr.2016.4928

Arora, A., Sharma, L., Sharma, D., Ghangale, G., Bidkar, J. and Tare, H., 2023. The nutraceutical role of pumpkin seed and its health effect: A review. Int. J. Pharma. Qual. Assur., 14: 233-238. https://doi.org/10.25258/ijpqa.14.1.40

Ayza, M.A., Zewdie, K.A., Yigzaw, E.F., Ayele, S.G., Tesfaye, B.A., Tafere, G.G. and Abrha, M.G., 2022. Potential protective effects of antioxidants against cyclophosphamide-induced nephrotoxicity. Int. J. Nephrol., pp. 5096825. https://doi.org/10.1155/2022/5096825

Badavi, M., Abedi, H.A., Dianat, M. and Sarkaki, A.R., 2013. Exercise training and grape seed extract co-administration improves lipid profile, weight loss, bradycardia, and hypotension of STZ-induced diabetic rats. Int. Cardiovasc. Res. J., 7(4): 111–117.

Bancroft, J.D. and Layton, C., 2019. The hematoxylin and eosin, In: (eds. S.K. Suvarna, C. Layton, and J.D. Bancroft), Bancroft’s theory and practice of histological techniques, 8th Ed. Elsevier, Philadelphia, pp. 126–138. https://doi.org/10.1016/B978-0-7020-6864-5.00010-4

Cetik Yildiz, S., Ayhanci, A. and Sahintürk, V., 2015. Protective effect of carvacrol against oxidative stress and heart injury in cyclophosphamide-induced cardiotoxicity in rat. Braz. Arch. Biol. Technol., 58: 569-576. https://doi.org/10.1590/S1516-8913201500022

Costello, L.C. and Franklin, R.B., 2016. A comprehensive review of the role of zinc in normal prostate function and metabolism; and its implications in prostate cancer. Arch. Biochem. Biophys., 611: 100-112. https://doi.org/10.1016/j.abb.2016.04.014

D’andrea, M., Lawrence, D., Nagele, R., Wang, C. and Damiano, B., 2008. PCNA indexing as a preclinical immunohistochemical biomarker for testicular toxicity. Biotech. Histochem., 83(5): 211-220. https://doi.org/10.1080/10520290802521804

Das, U.B., Mallick, M., Debnath, J.M. and Ghosh, D., 2002. Protective effect of ascorbic acid on cyclophosphamide-induced testicular gametogenic and androgenic disorders in male rats. Asian J. Androl., 4(3): 201-207.

Do Nascimento, C.C., Junior, O.A. and D’Almeida, V., 2020. Morphologic description of male reproductive accessory glands in a mouse model of mucopolysaccharidosis type I (MPS I). J. Mol. Histol., 51(2): 137–145. https://doi.org/10.1007/s10735-020-09864-x

Dowidar, M., Ahmed, A. and Mohamed, H., 2020. The critical nutraceutical role of pumpkin seeds in human and animal health: An updated review. Zagazig Vet. J., 48: 199-212. https://doi.org/10.21608/zvjz.2020.22530.1097

Fusco, R., Salinaro, A.T., Siracusa, R., D’amico, R., Impellizzeri, D., Scuto, M., Ontario, M.L., Crea, R., Cordaro, M., Cuzzocrea, S., Di Paola, R. and Calabrese, V., 2021. Hidrox® counteracts cyclophosphamide-induced male infertility through NRF2 pathways in a mouse model’, Antioxidants (Basel), 10(5): 778. https://doi.org/10.3390/antiox10050778

Ghafouri-Fard, S., Shoorei, H., Abak, A., Seify, M., Mohaqiq, M., Keshmir, F., Taheri, M. and Ayatollahi, S.A., 2021. Effects of chemotherapeutic agents on male germ cells and possible ameliorating impact of antioxidants. Biomed. Pharmacother., 142: 112040. https://doi.org/10.1016/j.biopha.2021.112040

Ghobadi, E., Moloudizargari, M., Asghari, M.H. and Abdollahi, M., 2017. The mechanisms of cyclophosphamide-induced testicular toxicity and the protective agents. Expert. Opin. Drug Metab. Toxicol., 13(5): 525-536. https://doi.org/10.1080/17425255.2017.1277205

Gu, Y.P., Yang, X.M., Luo, P., Li, Y.Q., Tao, Y.X., Duan, Z.H., Xiao, W., Zhang, D.Y. and Liu, H.Z., 2017. Inhibition of acrolein-induced autophagy and apoptosis by a glycosaminoglycan from Sepia esculenta ink in mouse Leydig cells. Carbohydr. Polym., 163: 270-279. https://doi.org/10.1016/j.carbpol.2017.01.081

Gupta, M., Dey, S., Marbaniang, D., Pal, P., Ray, S. and Mazumder, B., 2020. Grape seed extract having a potential health benefits. J. Food Sci. Technol., 57(4): 1205-1215. https://doi.org/10.1007/s13197-019-04113-w

Haghi-Aminjan, H., Asghari, M.H., Farhood, B., Rahimifard, M., Hashemi, G.N. and Abdollahi, M., 2017. The role of melatonin on chemotherapy-induced reproductive toxicity. J. Pharm. Pharmacol., 70(3): 291-306. https://doi.org/10.1111/jphp.12855

Hasseeb, M., Al-Hizab, F. and Hamouda, M.A.H., 2013. Impacts of grape seed oil supplementation against the acrylamide induced lesions in male genital organs of rats. Pak. Vet. J., 33: 282-286.

Hussien, N.A., Omara, E.A., El-Watidy, M.A. and El-Ghor, A.A., 2013. Chemotherapeutic potential of Grape Seed Extract (GSE) against experimentally induced precancerous stage in mice colon. J. Asian Sci. Res., 9: 2335-2346.

Iqubal, A., Syed, M.A., Najmi, A.K., Ali, J. and Haque, S.E., 2020. Ameliorative effect of nerolidol on cyclophosphamide-induced gonadal toxicity in Swiss Albino mice: Biochemical, histological and immunohistochemical based evidences. Andrologia, 52(4): e13535. https://doi.org/10.1111/and.13535

Kumar, V., Abbas, A.K. and Aster, J.C., 2017. Robbins basic pathology. E-Book, 10th ed, Elsevier Health Sciences.

Lestari, B. and Meiyanto, E., 2018. A review: The emerging nutraceutical potential of pumpkin seeds. Indones. J. Cancer Chemoprevent., 9: 92. https://doi.org/10.14499/indonesianjcanchemoprev9iss2pp92-101

Martins, R.V., Silva, A.M., Duarte, A.P., Socorro, S., Correia, S. and Maia, C.J., 2021. Natural products as protective agents for male fertility. BioChem., 1(3): 122-147. https://doi.org/10.3390/biochem1030011

Mohamadi, F., Nikzad, H., Taherian, A., Taghizadeh, M., Azami-Tameh, A., Naderian, H. and Atlasi, M.A., 2014. Combined effect of ginger and pumpkin seed extracts on rat testis and serum biochemical parameters after cyclophosphamide treatment. Anatomic. Sci. J., 11(1): 33-40.

Moosavi, F., Hosseini, R., Saso, L. and Firuzi, O., 2015. Modulation of neurotrophic signaling pathways by polyphenols. Drug Des. Dev. Ther., 10: 23-42. https://doi.org/10.2147/DDDT.S96936

Noda, T. and Ikawa, M., 2019. Physiological function of seminal vesicle secretions on male fecundity. Reprod. Med. Biol., 18(3): 241-246. https://doi.org/10.1002/rmb2.12282

O’Flaherty, C., 2019. Orchestrating the antioxidant defenses in the epididymis. Andrology, 7(5): 662-668. https://doi.org/10.1111/andr.12630

Oltra, B., Pozuelo, J., Rodríguez, R., Ingelmo, I., Arriazu, R. and Santamaría, L., 2014. Evaluation of PCNA, caspase 3 and E-cadherin on the ventral prostate of soy treated rats. J. Open Reprod. Sci. J., 6: 8-16. https://doi.org/10.2174/1874255601406010008

Pavin, N.F., Izaguirry, A.P., Soares, M.B., Spiazzi, C.C., Mendez, A.S.L., Leivas, F.G., Dos Santos Brum, D. and Cibin, F.W.S., 2018. Tribulus terrestris Protects against Male Reproductive Damage Induced by Cyclophosphamide in Mice. Oxid. Med. Cell Longev., pp. 5758191. https://doi.org/10.1155/2018/5758191

Richter, K. and Kietzmann, T., 2016. Reactive oxygen species and fibrosis: further evidence of a significant liaison. Cell Tissue Res., 365(3): 591-605. https://doi.org/10.1007/s00441-016-2445-3

Sabik, L.M. and Abd El-Rahman, S.S., 2009. Alpha-tocopherol and ginger are protective on Cyclophosphamide-induced gonadal toxicity in adult male albino rats. Basic Appl. Pathol., 2(1): 21-29. https://doi.org/10.1111/j.1755-9294.2009.01034.x

Shaban, A. and Sahu, R.P., 2017. Pumpkin seed oil: An alternative medicine. Int. J. Pharmacogn. Phytochem. Res., 9(2): 11. https://doi.org/10.25258/phyto.v9i2.8066

Stevenson, D.G., Eller, F.J., Wang, L., Jane, J.L., Wang, T. and Inglett, G.E., 2007. Oil and tocopherol content and composition of pumpkin seed oil in 12 cultivars. J. Agric. Food Chem., 55(10): 4005-4013. https://doi.org/10.1021/jf0706979

Thuan, D.T.B., Zayed, H., Eid, A.H., Abou-Saleh, H., Nasrallah, G.K., Mangoni, A.A. and Pintus, G.A., 2018. Potential link between oxidative stress and endothelial-to-mesenchymal transition in systemic sclerosis. Front. Immunol., 9: 1985. https://doi.org/10.3389/fimmu.2018.01985

Verze, P., Cai, T. and Lorenzetti, S., 2016. The role of the prostate in male fertility, health and disease. Nat. Rev. Urol., 13: 379–386. https://doi.org/10.1038/nrurol.2016.89

Wadi, S.A., Hameed, M.A., Sarhat, E.R., Sarhat, T.R. and Abass, K.S., 2021. Protective effect of pumpkin seed oil on methotrexate -induced nephrotoxicity in rats. Biochem. Cell. Arch., 21: 5383-5390.

Wang, F., Yang, W., Ouyang, S. and Yuan, S., 2020. The vehicle determines the destination: The significance of seminal plasma factors for male fertility. Int. J. Mol. Sci., 21(22): 8499. https://doi.org/10.3390/ijms21228499

Xanthopoulou, M.N., Nomikos, T., Fragopoulou, E. and Antonopoulou, S., 2009. Antioxidant and lipoxygenase inhibitory activities of pumpkin seed extracts. Food Res. Int., 42(5-6): 641-646. https://doi.org/10.1016/j.foodres.2009.02.003