The effects of ellagic acid on testicular tissue changes, sexual hormones, antioxidant system 1 and Gene Expression of Caspase-9 and Bcl-2 in the relative sterility rat model following 2 administration of busulfan: A stereological study 3

41 Background: Busulfan is an antineoplastic medication that is broadly utilized for cancer treatment. 42 On the other hand, prescription of busulfan may cause sterility in male patients. Therefore, the 43 decrease of this side effect is important. The aim of the present study was to evaluate the effects of 44 ellagic acid on testicular tissue changes, sexual hormones, antioxidant defense system, and caspase- 45 9 and Bcl2 gene expression in the relative sterility rat model following administration of busulfan. 46 Methods: Rats were randomly assigned to five groups of 13 animals per group. Sterility was 47 induced by a single injection of busulfan (10 mg/kg) in groups 3, 4 and 5. The control group was 48 not treated. The healthy group received 50mg/kg ellagic acid. Groups 4 and 5 (treatment group) 49 received 10mg/kg and 50mg/kg ellagic acid, respectively for 48 days. Then, the serum levels of 50 antioxidant enzymes, Malondialdehyde, sexual hormones and the testicular damage were 51 evaluated. 52 Results: The significant increment of total antioxidant capacity and catalase was seen in both 53 treatment groups (p<0.001). Also, both treatment groups significantly increased spermatogonia, 54 round spermatids and long spermatids. Treatment with 50mg/kg ellagic acid significantly 55 increased the testis weight, testis volume, seminiferous tubule volume, germinal epithelium 56 volume, interstitial tissue volume, spermatocyte, Sertoli cells, and Leydig cells in the busulfan 57 group(P<0.05). Additionally, 50mg/kg ellagic acid significantly increased the gene expression of 58 Bcl2 and decreased caspase 9 in the busulfan group (P<0.05). 59 Conclusions: The consumption of ellagic acid may have beneficial effects on antioxidant defense 60 system, sexual hormones abnormality and testicular tissue damage. 61 62 63 65 66

Background 87 Chemotherapy are associated with many changes in the reproductive system and among them, 88 alkylating agents cause the most adverse effects on the gonad [1,2]. Busulfan is one of the drugs 89 that has alkylating properties [3,4], and leads to enhanced oxidative stress, apoptosis, necrosis and 90 finally decreases the activity of the gonads and endocrine abnormality [5,6,1]. 91 According to studies, the fetus or neonate of rats that were born from pregnant mothers who have 92 been exposed to this drug during pregnancy had gonadal dysfunction and reduced testicular germ 93 cells and somatic cells [7,8]. 94 Administration of busulfan as a single dose in high doses (40-55 mg/kg body weight) in adult 95 mice induces azoospermia [9,10]. It has been shown that treatment with busulfan combined with 96 cyclophosphamide leads to enhanced oxidative stress, apoptosis, necrosis and finally decreases 97 the activity of the gonads and endocrine abnormality[5, 6, 1]. 98 Biological compounds with antioxidant properties such as ellagic acid with antioxidant properties 99 are able to protect the tissues against reactive oxygen species [11,12]. increases the activity of the 100 three antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, 101 which are altered in diseases caused by free radicals [13]. Studies have shown that ellagic acid has 102 beneficial neuroprotective effects against ischemic brain injury [12]. Therefore, according to these 103 findings, the aim of the present study was to investigate the effect of ellagic acid on the testicular 104 tissue changes, sexual hormones (testosterone, LH, and FSH), antioxidant defense system, and 105 caspase-9 and Bcl2 gene expression in the relative sterility rat model following administration of  five per cage, with 12:12 hours light-dark cycles at temperature of 23±2°c.

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Induction of relative sterility 114 The relative sterility rat model was induced by intraperitoneal administration of a single dose of 10 115 mg/kg busulfan (Pierre Fabre, France).

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Experimental design 117 The rats were divided randomly into five groups of 13 rats per group.

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Group 1, the control group, did not undergo any treatment and received only regular water and 119 food.

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The blood samples were centrifuged at 3500 rpm for 10 min to separate the serums and stored at -131 80°C prior to biochemical measurements. The sex hormones including testosterone, LH and FSH 132 were assessed by specific hormone kits (Bioassay Technology laboratory, China) and ELISA.  Stereological study 137 At the end of the assay, the left testicle tissue was separated from all the surrounding tissues; then, 138 the weight of the testicles was calculated by scales, and the primary volume was determined using 139 the immersion technique. In this study, "Orientator method" was used to acquire Isotropic uniform 140 random. In the next step, we put the slice testes in paraffin molds, so that the trocar fragment is   Where the "ΣPStructure" was the number of points hitting the profiles of the germinal epithelium or 152 tubules or interstitial tissue and "ΣP references" was the number of points hitting the testis.

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The method of calculation of numerical density and absolute number of cells [15-17] was as 154 follows: Where ΣQ was the number of the whole cells counted in all the dissectors, h was the height of the 157 optical dissector, a/f was the area of the counting frame, Σp was the total number of the counted 158 frames, BA was the microtome block advance to cut the block, and t was the mean of the final 159 section thickness.

RNA isolation and quantitative RT-PCR Gene expression levels 161
The total RNA from the testicular tissue was isolated using the TRIzol reagent (Invitrogen), and 162 the cDNA was synthesized following the manufacturer's protocol, using 1 μg RNA (Prime Script TM

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RT reagent Kit, Takara). RT-PCR was done using a standard SYBR-green PCR kit (SYBR Premix 164 EX Taq TM II, Takara), and the gene-specific PCR amplification was conducted using the Applied    These parameters significantly decreased in the BUS group more than the control group (P<0.05).

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The testis weight, testis volume, seminiferous tubule volume, germinal epithelium volume, seems that the improvement in spermatogenesis is due to the antioxidant activity of Ellagic acid.

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The study carried out by Motlag et al. showed that ellagic acid could prevent the reduction of 249 spermatogonia, Leydig and sertoli cells as well as diameter of spermatozoa tubules in the testicular 250 tissue of the rats exposed to cadmium chloride [24], which is similar to our results.

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Our major findings showed that ellagic acid potentially augmented the defense antioxidant

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It has been shown that Bcl-2 is a key factor in the inhibition of apoptosis; it is assumed that its The results demonstrated that the consumption of ellagic acid may have beneficial effects on 294 antioxidant defense system, sexual hormones abnormality and testicular tissue damage.

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Therefore, ellagic acid therapy may be effective in the treatment of reproductive defects caused 296 by chemotherapy.   of Helsinki and its later amendments.

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Competing interests 326 The authors declare that they have no competing interests. The results are presented as mean ± SD. There were no significant differences between the columns 434 containing at least one similar letter. However, different letters reveal a significant difference (p < 435 0.05).