|Year : 2020 | Volume
| Issue : 1 | Page : 6-11
Effects of pomegranate peel extract on the reproductive tract complications of sodium selenite in immature male rats
Fatemeh Namazi1, Mahboobeh Ashrafi2, Asghar Mogheiseh3, Esmaeil Bandariyan3, Arash Rakhshi-Asl2, Forough Zarei-Kordshouli1
1 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
2 Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
3 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
|Date of Submission||09-May-2020|
|Date of Decision||02-Nov-2020|
|Date of Acceptance||31-Dec-2020|
|Date of Web Publication||24-Aug-2021|
Dr. Asghar Mogheiseh
Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, P.O.Box 7144169155, Shiraz
Source of Support: None, Conflict of Interest: None
Background: Oxidative stress is one of the main causes of cataract and can be induced by selenite administration in rats. In experimentally induced cataract, sodium selenite may affect the reproductive system in young animals because of induction of oxidation condition. Methods: Sixty-four male 8-day-old Sprague Dawley rats were divided into five groups: control; pomegranate peel extract (PPE) 500 mg/kg; sodium selenite 30 μmol/kg; PPE and sodium selenite simultaneously on day 8; and PPE on day 6 and sodium selenite on day 8. A histopathological study was performed after 4 weeks. Results: Cataract was induced in all treated rats with selenite sodium. The volume and weight of the right and left testicles, the weight of the right and left epididymis, and the weight of the Cowper's gland, seminal vesicles, and prostate were significantly different between the sodium selenite group and other groups. On the other hand, the histopathological examination revealed tissue damage, interstitial edema, and the degeneration of spermatogenic cells in seminiferous tubules in the group which had received sodium selenite. There was a significant difference in all of the morphometric parameters of the genital system in rats, except the weight of the prostate between the PPE group and PPE and sodium selenite simultaneously group. Conclusion: The consumption of PPE improves the testis pathology indices in immature male rats with cataract induced by sodium selenite.
Keywords: Immature, male rats, pomegranate, reproduction, sodium selenite
|How to cite this article:|
Namazi F, Ashrafi M, Mogheiseh A, Bandariyan E, Rakhshi-Asl A, Zarei-Kordshouli F. Effects of pomegranate peel extract on the reproductive tract complications of sodium selenite in immature male rats. J Integr Nephrol Androl 2020;7:6-11
|How to cite this URL:|
Namazi F, Ashrafi M, Mogheiseh A, Bandariyan E, Rakhshi-Asl A, Zarei-Kordshouli F. Effects of pomegranate peel extract on the reproductive tract complications of sodium selenite in immature male rats. J Integr Nephrol Androl [serial online] 2020 [cited 2022 Aug 15];7:6-11. Available from: http://www.journal-ina.com/text.asp?2020/7/1/6/324503
| Introduction|| |
Cataracts are caused by factors such as aging, head trauma, long exposure to sunlight (ultraviolet radiation), diabetes, renal impairment, and oxidative stress. Oxidative stress is known as one of the most important factors in the onset of cataract. Selenium as an essential trace element is cotranslationally incorporated into proteins as selenocysteine. The selenoproteins have several properties such as antioxidant effect, anti-inflammatory activity, chemopreventive, and antiviral activity. However, selenium chemoprotective and antioxidant effects are limited to the low margin of safety (1–3 μg Se/g diet). The toxicity threshold of selenium is 3–4 μg Se/g diet. The ability of selenite to induce cataract in animal experiments was first reported in 1978. The mechanisms responsible for cataract induction by this substance include changes in lens epithelial cells metabolism, calcium accumulation, proteolysis induced by calpain enzyme, crystalline deposition, loss of cytoskeleton, and especially, oxidative stress. The administration of selenite causes lipid peroxidation, hydrogen peroxide formation, and glutathione (GSH) reduction in rats' lens. Cataract induced by selenite has many similarities with cataracts caused by aging in humans. This substance can cause cataract if given to rats before the age of 16 days.
Oxidative stress is a pathologic factor in the infertility of approximately half of men. This factor, which acts through two mechanisms, involves damage to the sperm membrane and consequently, reduces its mobility, causes damage to the sperm DNA, and finally, decreases the fertility rate which leads to a reduction in the pregnancy rate. Oral exposure of very high amounts of selenium (at least 8 times greater than those normally supplied by an adequate diet) has caused decreased sperm counts, increased abnormal sperm,, as well as testicular hypertrophy, degeneration and atrophy, changes in the female reproductive cycle in rabbits and rats, and changes in the menstrual cycle in monkeys., To the best of our knowledge, we could not find any report about the toxicity of subcutaneous injection of selenium on the development of immature male reproductive tract in rats.
Pomegranate peel extract (PPE) has high antimutagenic, antioxidant, antimicrobial, and antiapoptotic properties due to its significant amounts of polyphenols. Therefore, considering the important role of oxidative stress in the development of cataract and subsequent fertility decline following exposure to high dose of selenite sodium and the antioxidant properties of aqueous extract of pomegranate peel, this study was aimed to investigate the toxicity of high dose of selenite sodium and protective effects of PPE on the reproductive tract of immature male rats.
| Materials and Methods|| |
Statement of animal rights
In this research, the principles of working with laboratory animals were carried out in accordance with the bioethics code of the School of Veterinary Medicine, Shiraz University, Shiraz (IACUC no: 4687/63). The recommendations of the European Council Directive (2010/63/EU) of September 22, 2010, regarding the standards in the protection of animals used for experimental purposes, were also followed.
Preparation of aqueous pomegranate peel extract
Fresh pomegranate fruits were collected from the Faroogh region around of Shiraz, Iran, during the fall season. Then, the internal peels were separated manually and shade-dried for 3 days. The dried pomegranate peels were grinded in distilled water for 24 h; the pomegranate peels were soaked. And after filtration, the peels were dried by the freeze dryer and kept in the refrigerator until use. At the time of administration, after weighing the rats and calculating the required amount, the extract was dissolved in sterile distilled water and administered.
Animals and experimental design
Cataract was induced by sodium selenite and treated with the aqueous extract of pomegranate peel. Sixty-four 8-day-old male Sprague Dawley rats were divided into five groups in a randomized controlled trial: Group 1 (control, n = 8), Group 2 (recipient of PPE orally (500 mg/kg), twice a week for 4 consecutive weeks, n = 14)), Group 3 (received a single subcutaneous injection of sodium selenite on day 8 [30 μmol/kg;] n = 14), Group 4 (recipient of PPE and sodium selenite simultaneously on day 8, n = 14), and Group 5 (recipient of PPE on day 6 and sodium selenite on day 8, n = 14). Morphological examination of both eyes in each rat pup from all experimental groups was performed twice a week during the study by slit-lamp illumination and eye lens opacity was considered as the incidence of cataract. Rats were examined twice a week for cataract; after 4 weeks, rats were euthanized by human use of CO2.
Evaluation of reproductive parameters
In order to assess the reproductive system, testis, epididymis, and prostate gland, seminal vesicles and Cowper's gland were weighed after the separation and compared between groups. For the histopathological study, samples of testes in 10% neutral buffered formalin were sectioned at 5 μm and stained with hematoxylin and eosin. Histopathological changes were recorded and scored. Scoring of severity of histopathological lesions was: 0 = no specific pathologic lesions; 1 = mild lesions; 2 = moderate lesions, and 3 = severe lesions. Testicular volumes were measured by an electronic clipper of 900V model veterinary ultrasound.
Data were reported as mean ± standard deviation. They were analyzed by SPSS software Version 26.0 (IBM Corp, Armonk, NY), one-way analysis of variance, and appropriate post hoc test. P < 0.05 was considered as the significance level for all comparisons.
| Results|| |
The cataract was induced experimentally with injection of selenite sodium (30 μmol/kg/SC) in rats of groups 3–5. The incidence of cataract in the mentioned groups was 100%, albeit with different degrees.
Reproductive tract measurements
There was a significant difference in the body weight of rats (P = 0.0001) and left testicular weight (P = 0.04) between the sodium selenite group and the group that had received sodium selenite and PPE at the same time while there was only a significant difference in the body weight of the rats (P = 0.0001) between the sodium selenite group and the group which had received sodium selenite and PPE separately. No significant difference was observed in prostate weight among groups. The results of the statistical analysis and the comparison of data related to the measurements of different parts of the reproductive tract are presented in [Table 1]. In all the parameters related to the morphometry of the genital system, except the prostate weight (P = 0.96), there was a significant difference between the PPE group and the group which had received sodium selenite and PEE simultaneously. A significant difference was observed between the group which had received PPE and the group of PPE and sodium selenite at different days in all parameters except left testis volume (P = 0.2), prostate (P = 0.99), and seminal vesicles weight (P = 0.11). In the case of right (P = 0.0001) and left epididymal (P = 0.003) and seminal vesicles weight (P = 0.01), there was a significant difference between PPE and selenite groups. The control group showed a significant difference with the group which had received simultaneous PPE and sodium selenite in the left testis volume (P = 0.0006). There was a significant difference in the case of body weight (P = 0.0001), right epididymis weight (P = 0.0001), and right (P = 0.001) and left testis weights (P = 0.0001) between the control group and the group which had received sodium selenite and PPE simultaneously. Furthermore, the body weight of rats (P = 0.0001), right epididymis weight (P = 0.0001), and right (P = 0.03) and left testis weight (P = 0.0001) were significantly different between the control group and the group which had received sodium selenite and PPE separately. Cowper's gland (P = 0.04) and right epididymal (P = 0.0001) weight were significantly different between the control group and the group which had received PPE and sodium selenite separately. The only significant difference between the control group and sodium selenite was in the right epididymal weight (P = 0.0001). A significant difference was observed in the weight of the seminal vesicle between the control group and the group which had received PPE and sodium selenite simultaneously (P = 0.03).
|Table 1: Measurement and comparison of weight and volume of the testis, epididymis, Cowper, prostate, and vesicle seminal glands and weight of rats in immature rat treated with selenite sodium and aqueous extract of pomegranate peels after 1 month|
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No lesions were observed in the tissue sections of the control and extract groups [Figure 1]a, whereas the rats receiving selenite showed mild to moderate interstitial edema, moderate degeneration of spermatogenic cells, and a decrease of sperm in the seminiferous tubules [Figure 1]b. Similar lesions were also seen in the tissue sections of the rats which had received the extract before selenite [Figure 1]c. The tissue sections of the rats which had received extract and selenite simultaneously revealed mild edema and mild degeneration of spermatogenic cells [Figure 1]d. These alterations were statistically significant between the last group and those which had received selenite [Table 2].
|Table 2: Histopathological evaluation and scoring of edema and degeneration (mean±standard deviation) in the testis are presented after 1 month of treatment of immature male rats with selenite sodium and/or aqueous extract of pomegranate peels|
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|Figure 1: Histopathological evaluation of the testis tissue was performed at the end of the study and after treated immature male rats with PPEs and/or sodium selenite. (a) PPE extract group: Tissue sections showed intact structure, H and E, Scale bar = 100 μm; (b) Sodium selenite group: Tissue sections showed moderate interstitial edema (*) with moderate degeneration of spermatogenic cells (arrow) as moderately decrease and loss of these cells, H and E, Scale bar = 250 μm; (c) PPE extract and sodium selenite separately group: lesions were similar to selenite group, H and E, scale bar = 250 μm; (d) Sodium selenite + PPE extract group: tissue sections showed mild edema (*) with mild degeneration of spermatogenic cells (arrow) as mildly decrease and loss of these cells, H and E, scale bar = 250 μm. PPE: Pomegranate peel extract|
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| Discussion|| |
In the present study, the results showed that the administration of sodium selenite had negative effects on the weight and volume of the testis, epididymis, and accessory sex glands and histological indices of testis in immature male rats. PPE administration, simultaneously or before the injection of sodium selenite, significantly reduced the sodium selenite negative effects.
The single injection of sodium selenite (30 μmol/kg/SC) induced cataract in all rats with different degrees. The incidence of the cataract has been studied with subcutaneous administration of sodium selenite to 10-day-old male Wistar rats at doses of 5, 10, 20, 40, and 60 μmol/kg of body weight. At the end of the study (20 days), except cataract, no symptoms of other diseases were observed. Their findings were inconsistent with the findings of the present study. Injuries depended on the amount of prescribed sodium selenite, and doses of 20 and 40 μmol/kg had the greatest effect on permanent cataract formation. The difference between the results of this study and early mentioned study may be related to the duration of the experiment. The zinc sulfate and sodium selenite supplements were used to counteract the gamogentic and steroidogenic diseases caused by long swimming stress. Based on the results, these compounds reduced oxidative stress and improved the reproductive parameters in male mice. Furthermore, the difference observed between the findings of the present study and other studies is probably due to the age of mice (other researchers used 140-day-old mice in their experiment). In addition, the discrepant results could be attributed to the administration of zinc supplementation with sodium selenite simultaneously. In another study, researchers reported the favorable effects of selenium and sodium selenite nanoparticles on the reproductive performance in male mice. A significant difference was observed in all parameters including sperm parameters, oxidative stress biomarkers, and histology between the control and treatment groups. During the study, the antioxidant capacity of the treated group with selenium nanoparticles was higher than the control group, but the sperm quality and the histological characteristics indicated higher antioxidant effects of nanoparticles than sodium selenite during a spermatogenesis cycle. In their study, the mice were mature and sodium selenite may exhibit antioxidant properties at this age. The protective effects of sodium selenite on the reproductive damage induced by methyl parathion in male rats have been documented. Methyl parathion at a dose of 0.28 mg/kg caused body and testicular weight loss, reduced the diameter of the seminiferous tubules, plasma levels of LH, and testosterone, and increased the intercellular space and damage to the tissue necrosis. The administration of sodium selenite improved the fertility parameters in the rats which had received methyl parathion. Differences observed in the results may be due to higher levels of sodium selenite administered in our study. The protective effects of sodium selenite have been evaluated on the diabetic and nondiabetic rats that were damaged by lead nitrate. The evaluation of the results after 4 weeks of treatment did not indicate a significant difference between the control group and the treated group with sodium selenite in the parameters of malondialdehyde, superoxide dismutase, catalase, GSH peroxidase, and GSH S-transferase while these parameters increased in the diabetic and nondiabetic rats following the administration of lead nitrate. Furthermore, lead nitrate caused histopathological changes in the testicular tissue of rats. Both groups, diabetic and nondiabetic rats receiving sodium selenite combined with lead nitrate, showed a significant decrease in malondialdehyde and superoxide dismutase levels with mild pathological changes. Therefore, it was found that sodium selenite could significantly reduce the negative effects of lead nitrate on fertility in diabetic and non-diabetic male rats. It seems that at a higher age and in situations in which distress and disease are present, sodium selenite exhibits antioxidant properties and thus, it can have protective effects. The effects of sodium selenite have been studied on testicular tissue damages that caused by cisplatin in adult male rats. The results indicated that the body and testicular weight decreased in cisplatin-treated rats. Furthermore, the administration of this drug caused damage to the seminiferous tubules and diminished the process of spermatogenesis. Sodium selenite consumption reduced the fertility damage in male rats following the administration of cisplatin.
The subcutaneous injection of sodium selenite (30 μmol/kg) in our study decreased the weight of the left and right epididymis and increased interstitial edema and degeneration of spermatogenic cells in compared with rats in control group. The oral administration of high levels of selenium in rabbit (0.001 mg/kg/day) and Wistar rat (0.234 mg/kg/day) has adverse effects on testosterone concentration and sperm quality and induced testicular atrophy,, but the harmful effect of selenite intake was not observed on sperm quality in mice (3.31–5.45 mg/kg/day). The decreasing quality of semen has been a dose-dependent effect in the wild-caught rat (0.1–0.2 mg/kg/day). Likely, administration of high levels of sodium selenite destroys interstitial and spermatogenic tissues of the testis and subsequently decreased the level of testosterone and semen quality. Furthermore, the adverse effect of sodium selenite on epididymis could additionally decrease sperm quality and fertility.
To the best of our knowledge, no study has so far investigated the protective and therapeutic effects of PPE on the reproductive tract in rats with induced cataract caused by sodium selenite. In our study, the significant and lower volume and weight of testes, weight of epididymis, vesicular glands, and weight of rats were observed in rats received the combination of PPE extract and sodium selenite in compared with PPE extract. The administration of PPE extract alone and in combination with selenite sodium could decrease interstitial edema and degeneration of spermatogenic cells in comparison with control and selenite groups respectively. Some studies have reported the antioxidant effects of peel, leaves, and the seed extract of pomegranate. The effects of pomegranate juice and methanolic PPE have been evaluated on the reproduction of adult albino Wistar rats. The results indicated the high antioxidant properties of pomegranate juice and its methanolic extract. These supplements may increase the serum levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). In a similar study performed by other researchers, a significant increase was observed in the testicular weight. It has been determined that the methanolic extract of peel has the highest antioxidant activity among all the extracts of different parts of pomegranate., Pomegranate juice also improves the quality of sperm, spermatogenic cell density, epididymal sperm concentration, and sperm motility. The pomegranate juice increased the serum levels of testosterone, LH, and FSH in rats treated with carbon tetrachloride. Furthermore, researchers found that the degeneration rate of Leydig cells and germ cells that cause spermatozoa disorder decreased due to the use of pomegranate juice. The positive protective effects of pomegranate juice were studied on the reproductive hormones of adult male rats after the administration of acrylamide. Acrylamide consumption significantly decreased testosterone levels, increased levels of FSH and LH, reduced spermatozoa concentration, and increased spermatic abnormalities.
| Conclusions|| |
The adverse effects of sodium selenite were observed in the reproductive tract of immature rats. The administration of PPE separately and simultaneously with sodium selenite was found to reduce the histopathological lesions and improve testis, epididymis, and accessory sex glands' volume and weight of Sprague Dawley immature male rats.
Financial support and sponsorship
This study was supported financially by the School of Veterinary Medicine, Shiraz University (grant number 96GCU2M154630). There was no significant financial support for this work that could have influenced its outcome. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]