International Medical Journal Vol. 27, No. 5, pp. 545 - 548 , October 2020

DIABETES MELLITUS

Protective Effects of Zinc Supplement on Chromatin Deficiency and Sperm Parameters in Streptozotocin-Induced

Diabetic Rats

Hakimeh akbari1,3), Hosein Forouzandeh1,2), Mehdi Ghavamizadeh1,3)

ABSTRACTObjective: Diabetes mellitus (DM) is one of the metabolic diseases that may effect on sperm's health and male reproductive

function. Also zinc supplement had many antioxidant agents that may improve adverse effects of diabetes on DNA integrity and sperm chromatins.

Materials and methods: In this study 36 Wistar Rat (200 gr) were randomly divided non-diabetic and Streptozotocin-induced diabetic, then were divided into 4 groups (n = 9): zinc sulfate (25 mg daily) and sailin administration. Then sperm parameters and semen analysis were assessed, thirty-five days later according to World Health Organization (WHO) standards. The sperm DNA fragmentation was measured by sperm chromatin dispersion (SCD) test, protamine deficiency was determined by chromo-mycin A3 (CMA3) staining.

Results: The sperm count and viability increased in zinc supplement groups, also head and neck deformity were seen less than saline administration groups(p < 0.05). In Zinc administration groups, the sperm motility type C, D reduced; furthermore abnormal morphology, DNA fragmentation and protamine deficiency over than 30% were reduced (p < 0.05).

Conclusion: The results show the protective effects of Zinc supplement on sperm parameters, chromatin efficiency and DNA integrity caused by diabetes. The beneficial effect of Zinc supplement might be associated to the inhibition of oxidative stress accordingly a favorable antioxidant therapeutic candidate for the management of male infertility.

KEY WORDS diabetes, sperm, infertility, rat, chromatin

Received on December 22, 2019 and accepted on March 2, 20201) Cellular and Molecular Research Center, Gerash University of Medical Sciences 3) Student Research Committee, Gerash University of Medical Sciences Gerash, Iran Gerash, Iran2) Blood Transfusion Research Center, High Institute for Research and Correspondence to: Hossein Forouzandeh Education in Transfusion Medicine (e-mail: hosainforuozandeh@yahoo.com) Shiraz, Iran

545

INTRODUCTION

Infertility is defined as the inability to have children after at least one year of marriage without the use of any contraceptives methods1). About 40-50% of human infertility caused by male factors due to deficiencies in the semen, then semen analysis is the main criteria for male fecundi-ty2). Researchers had shown that negative correlation between percentag-es of spermatozoa health's and levels of reactive oxygen species (ROS) in seminal fluid; The level of seminal ROS is regulated by seminal pro-teins, vitamins, enzyme and non-enzymatic antioxidants. so these factors have beneficial effects on sperm parameters and developmental capaci-ty3,4). Diabetes mellitus (DM) is a chronic metabolic disorder associated with ROS and other metabolic problems such as disruption of proteins, carbohydrates and lipids metabolism; can alter the structure of sperm chromatin5). In diabetic patients hyperglycemia reduces serum levels of LH, FSH and testosterone, that consequently affected sperm quality and their fertility potential6). Hyperglycemia increases the levels of ROS trough over generation of superoxide in mitochondria, accordingly the body's antioxidant capacity is decreased7,8). In the normal condition there is a balance between the antioxidant activity and ROS production in male reproductive system by scavenging or removing the ROS9). The level of abnormal sperm morphology and DNA fragmentation in diabetic men is higher than normal; subsequently affect male fertility10). The endogenous antioxidant of seminal plasma protects the spermatozoa

from oxidative damage; however, can't prevent acrosome membranes and sperm tail from lipid peroxidation consequently the sperm cell needs extra antioxidant for protections11).

Zinc (Zn) is metalloproteinase cofactors that play an important role in scavenging ROS, DNA transcription, repair of damaged DNA and protein synthesis. In human seminal plasma, the concentration of zinc is higher than other tissues12). Many studies in asthenozoospermic patients have indicated the administration of zinc, vitamin E and vitamin C reduces the level of oxidative stress, sperm DNA fragmentation and apoptosis also increases the sperm count and motility13,14). The antioxidant therapy can progress the sperm quality and male fertility. As the antioxidant proper-ties of zinc sulfate could act against reproductive system impairment and the high rate of sperm disorders in diabetic patient, we interested to inves-tigate the zinc supplement protective effects on rat sperm parameters, against oxidative stress and tissue damages in diabetic rat.

MATERIALS AND METHODS

Chemicals phosphate-buffered saline (PBS), Tris (hydroxymethyl)aminometh-

ane, Dithiothreitol (DTT), Sodium dodecyl sulfate (SDS), Ethylene diamine tetraacetic acid (EDTA) , borate and Chromomycin A3 (CMA3)

C 2020 Japan Health Sciences University & Japan International Cultural Exchange Foundation

Akbari H. et al.546

were purchased from Sigma-Aldrich chemical company (St Louis, MO, USA). All chemicals and reagents used were analytical grade.

AnimalsIn this study, 36 Adult (5 weeks old) male Wistar rats (200 ± 20g)

were obtained from the animal house and research center of Gerash University of Medical Sciences, Gerash, Iran. After weighting, the rats were caged in distinct group (n = 9) at animal house with standard air flow and light-dark cycle was adjusted on 12h. Relative humidity and mean temperature was about (40-50 %) and (22-24℃) respectively and the rat was fed with water and food available; the experiment was approved by the institutional Animal Ethics Committee of Gerash University of medical science with referee number[IR.GERUMS 1396-1070].

Study designThe rats were divided into two groups: diabetic induced by

Streptozotocin [received a single dose of STZ (150 mg/Kg) intra-perito-

neal]15) and non-diabetic groups. 72 h later the rats were investigated for DM, if fasting serum glucose level were higher than 200 mg/dl were considered as diabetic16). Each group were randomly allocated into two subgroups by administration normal saline orally (400 μl Kg) or zinc sulfate supplement orally (10 mg/kg), once daily for 42 days, then sperm parameters were measured within four groups after this course. All rats were euthanized by cervical dislocation, then immediately blood was collected from the heart and centrifuged (3000 rpm) at 4℃ for 7 min using a micro centrifuge to separate the plasma from the blood cells and kept at -20℃ for evaluation of testosterone levels through using an ELISA kit (IBL Company, Japan)17). After euthanasia, testis, epididymis and vas deferens were measured and weighted. The right testis, caudal epididymis and vas deferens were removed for evaluation of sperm parameters then were gently operated and transferred into 2.5ccHam's F10 medium supplemented with 8 mg/ml bovine serum albumin [(BSA) Sigma Company, St. Louis, USA)], After 30 minute incubation at (37℃ and 5%CO2) in the humidified air, the sperm motility was assessed under a light microscope (Nikon TS100, Tokyo, Japan) via using a 400x magnification and almost 200 spermatozoa were evaluated for each specimen and reported as WHO protocols:

A (Direct path fast progressive sperm motility), B (tortuous path

Figure 1. Effect of zinc sulfate administration on blood testosterone level, the vital sperm and weight of rats, in diabetic and non-dia-betic Wistar male Rat. Data were analyzed by one-way ANOVA test followed by Tukey's post hoc test.

*significantly different from the control group (*P < 0.05). # significantly different from the zinc sulfate administration group (#P < 0.05).

Figure 2. Effect of zinc sulfate administration on types of sperm's deformity and protamin deficiency, in diabetic and non-diabetic Wistar male Rat. Data were analyzed by one-way ANOVA test followed by Tukey's post hoc test.

*significantly different from the control group (*P< 0.05). # significantly different from the zinc sulfate administration group (#P

< 0.05).

Figure 3. Effect of zinc sulfate administration on types of sperm's motility, in diabetic and non-diabetic Wistar male Rat. Data were analyzed by one-way ANOVA test followed by Tukey's post hoc test.

*significantly different from the control group (*P < 0.05). # significantly different from the zinc sulfate administration group

(#P < 0.05).

Protective Effect of Zinc Supplement in Diabetic Rats 547

fast progressive sperm motility).C (slow progressive sperm), D (immo-tile sperm) per total sperms.

The morphological sperm parameters evaluated as deformity in head, neck and tail.

Sperm Countimmediately after dissection the epididymis sperm fluid was dipped

and fixed by formalin/ saline then counted using an improved Neubauer counting chamber. We don sperm counted in four large squares as mean was multiplied by 104; finally, the result was described as the sperm's number in milliliter.

Sperm viability was assessed using the eosin stain then at least 100 spermatozoa were counted. The staining was done with a (10 μl) of sperm suspension and (20 μl) of eosin stain. Dead sperm cells were stained because of damaged membranes and sperm head seemed purple to red but the live sperms were unstained18).

The width, thickness and length also the volume of testis were mea-sured by using a standard digital caliper. The histological parameter such as thickness of germinal epithelium and diameter of seminiferous tubules were measured by a light microscope.

DNA fragmentation assay (SCD)sperm DNA integrity was assessed SCD test according the

Evenson's (2016) methods; briefly, the sperm chromatin and DNA were denatured in an acidic micro-gel base. After eliminating the proteins in chromatin, the DNA strands spread to the extent that possible around the sperm's head as a visible halo during staining. But with Sperm's DNA dispersion, around the sperm's head seemed very small halo or don'ts visible of halo caused by disruptions of DNA strands19).

Protamine Deficiency AssessmentProtamine deficiency was evaluated using CMA3 staining according

the Fortes's (2014) methods20). After semen samples were washed with PBS, the sample centrifuged 3 times (3000 rpm for 5 min). The fixa-tions carnous solution was added to the washed sperms and placed in refrigerator (4℃ for 5 min). These sample was spread onto glass slides and stained with 100 μl CMA3 (0.25%) in a dark room for 2 min. after the slides were washed and mounted, almost 200 sperms per slide were evaluated with fluorescence microscope (Japan-Nikon-Eclipse600)

under filter (460-470 nm) and magnification (100 X) to reporting posi-tive and negative CMA3 sperms.

Statistical analysisData were expressed as Mean ± SD and analyzed by the statistical

package SPSS software version 21 for Windows. Analysis was carried out using a One-way ANOVA followed by Tukey's post hoc test for comparison of data between these groups. Statistical significance was set at p < 0.05.

RESULTS

Serum Testosterone LevelThe blood testosterone level in diabetic rat decreased compared

with the non-diabetics, also increased this level in zinc sulfate adminis-tration group (Figure 1). Also the vital sperm significantly increased within no diabetic zinc sulfate administration group; but the weight of rats not significantly different between these groups.

Morphological studyThe number of Leydig cells was increased in zinc sulfate groups

and the most number was seen in no diabetic zinc sulfate administration. In zinc groups, the thickness of germinal epithelium and seminiferous tubules were higher than other groups.

The absolute weights of seminal vesicle, epididymis with vas defer-ens and testis in diabetic group were significantly lower, while in zinc supplement group, measured higher in comparison with control. In dia-betic groups showed an increase atrophy of seminiferous tubular and interstitial space rather than no diabetic but in diabetic-zinc the seminif-erous tubular was thicker. In zinc sulfate groups increased the normal morphology and reduction in tail deformity (p < 0.05) (figure 2).

Also, in zinc supplement group the sperm motility type A, B was higher than other groups and DNA integrity were higher than control group proved by the existence of high level in SCD1 and SCD2 within zinc supplement group while the SCD3 and SCD4 were less than groups (Figure 4).

DISCUSSION

In the present study, we assessed the sperm quality and testicular parameters in STZ-induced diabetic rat after zinc supplement administra-tion. In the current study the sperm viability, motility and number of health DNA integrity significantly decreased in diabetic rat, this was in agreement with Mangoli et al. Also they showed that chromatin integrity remained unchanged in the diabetic mice16). These differences with our result may be caused by differences in the sperm structure in rat with mice; also Yaghobi et al. showed that sperm parameters and testicular features non significantly decreased in STZ-induced diabetic mice that received saline compared with the intact animals21); while Ballester et al. have shown that serum testosterone level in STZ-induced diabetic rats significantly reduced rather than control animals22); in agreement with ours Nasrolahi et al. have shown that in diabetic rat the seminiferous tubules and sperm parameter was less than normal groups23). Guneli et al. was found to be disrupted the seminiferous tubule structure and decreased the spermatogenic cell in the diabetic rats24). In the present study, we considered a significant decrease in the seminiferous tubules diameter in diabetic rats compared with control which was in agreement with Atlay et al25). In contrast Salimnejad et al. have found that increased the diameter of seminiferous tubules in diabetic rats26). The seminiferous tubule structure in the diabetic rats was found to be disrupted, and there was a considerable decrease in the spermatogenic cell series. In addition, Padr´on et al27) by analyzing semen samples from healthy and diabetic subjects sperm parameter and spermatogenic function is related to Sertoli cells activity but Leydig cells that produce the testosterone hor-mone is related to steroidogenic dysfunction might be an indirect or direct consequence of insulin deficiency, furthermore, Xu et al. in dia-betic rats established that insulin additional inhibited reduction of testos-terone level, nevertheless did not affect other sperm parameters28).

The current study confirmed that diabetes mellitus caused testicular dysfunctions, in male rats and zinc sulfate administration improved the impairment of the spermatogenic cell series, the seminiferous tubules and sperm parameters such as motility and count. The number of DNA

Figure 4. Effect of zinc sulfate administration on the types of SCD, in diabetic and non-diabetic Wistar male Rat. Data were analyzed by one-way ANOVA test followed by Tukey's post hoc test.

*significantly different from the control group (*P < 0.05). # significantly different from the zinc sulfate administration group (#P

< 0.05).

Akbari H. et al.548

fragmentation and protamine deficiency was reduced after zinc sulfate administration in diabetic rats; this may be due to the zinc's antioxidant role. The generation of reactive oxygen species (ROS) is the most com-mon consequence of diabetes can induce cell failure and disturb the male reproductive function; furthermore induces an adverse change in chromatins quality and sperm parameters29,30). Zn is reported to play an important role in glucose homeostasis by enhancing hepatic glycogene-sis and improves glucose utilization31). Sperm analysis is valuable index for determination of the male infertility reason; many factors including luteinizing hormone (LH), follicle-stimulating hormone (FSH) and tes-tosterone affect male fertility32).The Reduce sperm count be affected by Short-term hyperglycemia, while prolonged hyperglycemia caused adverse effects on sperm count and motility through oxidative stress in diabetic rats33). Ding et al. described that the serum testosterone level in diabetic rats was lower rather than in control non diabetic rats, in agree-ment with our findings34). Zn increased the testosterone production by increased the release of FSH and LH from the pituitary gland, moreover inhibits the aromatase enzyme activity35). The oxidative stress reduces the leydig cells by enzymatic and non-enzymatic manner and causes reduction of testosterone level36). On the other hands, there is a negative correlation between normal spermatogenesis and increased ROS level37). The normal spermatogenesis depends on increased Leydig and Sertoli cells endocrine activity and decreased oxidative stress, and then accord-ing to this study, we can conclude that zinc sulfate can promote testos-terone biosynthesis, sperm quality and decrease tubular atrophy with antioxidants components by down-regulating oxidative stress.

CONCLUSION

The overproduction of ROS in seminal plasma in diabetic's cases, can affect sperm quality, testicular activity or seminal antioxidant defense mechanisms and cause oxidative stress, accordingly antioxidant therapy can improve the sperm chromatin deficiency and male fertility. The present study confirmed that zinc sulfate administration, enhanced sperm fertility characteristics and reduction DNA fragmentation, these protective effects may be due to the antioxidant effects and eliminating free radicals.

ACKNOWLEDGEMENTS

This research was financially supported by Ministry of Sciences and carried out at Gerash University of medical sciences.

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