Protective effect of vitamin E on sperm parameters, chromatin quality, and DNA fragmentation in mice treated with different doses of ethanol: An experimental study

Abstract Background Excessive consumption of alcohol induces an increase in oxidative stress production and can lead to detrimental effects on the male reproductive system. Objective To evaluate the possible protective effects of coadministration of vitamin (vit) E on the detrimental changes in the sperm quality of mice administered ethanol. Materials and Methods Fifty-four BALB/c mice were categorized into nine groups (n = 6/each). The control group received a basal diet while the eight experimental groups received ethanol 10%; ethanol 20%; vit. E 100 mg; vit. E 200 mg; ethanol 10% + vit. E 100 mg; ethanol 10% + vit. E 200 mg; ethanol 20% + vit. E 100 mg; ethanol 20% + vit. E 200 mg. After 35 days, the sperm parameters and sperm chromatin were assessed. Results The results demonstrated a significant reduction in the motility rate, normal morphology rate, viability rate, increase in abnormal DNA structure and packaging (TB staining), and DNA damage (TUNEL) in ethanol consumer groups. In addition, the findings showed a significant increase in the aforementioned parameters in ethanol- and vit. E-consumer groups compared to the ethanol-only consumer groups. The ethanol group received 20% of the most damage among the groups. The group receiving vit. E 100 mg and those receiving ethanol 10% + vit. E 200 mg gained the highest benefit among the groups. Conclusion Sperm forward progressive motility, normal morphology rate, and viability decreased in the ethanol groups. Also, the rates of spermatozoa with abnormal DNA structure and DNA fragmentation increased in the ethanol groups. Our findings revealed that the coadministration of vit. E and ethanol can protect destructive changes in DNA structure and damage.


Introduction
The American Society of Reproductive Medicine defines infertility as failure to achieve pregnancy after 1 yr timed unprotected intercourse (1). Approximately 15% of couples in the world suffer from infertility and 50% of cases are related to male causes (2). Many factors cause infertility, among them, ethanol, as a widely used drug, is well-known for suppressing reproductive function. Excessive consumption of ethanol is a significant public health problem.
Excessive consumption of alcohol results in a variety of pathological changes in male reproduction such as low sperm count, reduced motility and quality of spermatogenesis, altered testicular histology, and changes in sperm morphology such as breakage of the sperm head, distention of the mid-section, and tail curling in men and experimental animals (3).
It has been suggested that the mechanisms associated with acute and chronic alcohol consumption and decreased sperm quality are related to the production of reactive oxygen species (ROS) and reduction of nicotinamide adenine dinucleotide, which enhances the activity of the respiratory chain, ROS formation, and It also affects the metabolism of male hormones such as testosterone and subsequently has an adverse effect on the process of spermatogenesis (4). In addition, one of the products produced in the process of alcohol metabolism is acetaldehyde, interacts with proteins and lipids to produces ROS (5) ROS targets and damages plasma membranes and DNA molecules in sperm and other cells (6). High levels of ROS such as superoxide ions, hydrogen peroxide, peroxynitrates cause damage to cell components such as membrane lipids, proteins, organelles, and cell DNA. In addition, Damage caused by oxidative stress can also affect gene expression (7,8). A cohort study examining the effect of alcohol consumption in infertile individuals showed that alcohol consumption for 4-7 wk has no negative effect on sperm parameters (9).
Vit. E (α-tocopherol) is a fat-soluble organic material and the primary antioxidant component of the spermatozoa that is beneficial for the maintenance of mammalian spermatogenesis (11). "Vit. E in the form of α-tocopherol, it is a major antioxidant located within biological membranes that play a role in protecting from lipid peroxidation. α-tocopherol breaks the chain reactions of lipid peroxidation through the mechanism of donation of a hydrogen atom from its phenolic hydroxyl group to lipid peroxyl radical resulting in the formation of stable lipid hydroperoxide and unreactive tocopheroxyl radicals" (10). This vitamin has strong antioxidant properties, inhibits lipid peroxidation, and is a protector against the ROS in the testes.
A deficiency of vit. E leads to increased OS levels which are incompatible with normal spermatogenesis and testosterone production (12).
These observations strongly suggest that vit. E Maintains fertility of male by reducing oxidative stress. However, so far, the capacity of vit. E to prevent ethanol-induced toxicity in the reproductive system has not been determined.
The beneficial effects were evident in the form of increase in testes weight, semen-quality parameters, antioxidants status, and testosterone in mammals as well as birds (13). Vitamin E increases the level of antioxidants in the body and reduces the effect of oxidative damage on the testicles and increases sperm motility (14).
Hence, this study was designed to investigate the probable protective effects of coadministration of vit. E and different doses of ethanol on the semen parameters and sperm DNA integrity of BALB/c mice.

Animals and experimental design
In this experimental study, 54 healthy BALB/c mice (8-wk old, weighing 25 ± 2 gr) were

Epididymal sperm preparation
After 35 days, all mice underwent surgery and small pieces of epididymis were dissected and transferred into a 1-mL pre-warmed Ham's F10 medium (37°C, 5% CO 2 ). The epididymal tissue was then gently cut with a needle to allow the sperm to swim into the medium and then placed in the incubator for 15 min.

Sperm analysis
All protocols were done according to the National Institute of Health Guide for the care and use of laboratory animals.

Diff-Quick staining
Slides were stained with Diff-Quick staining.
The dried colored slides were scanned at 100× magnification to look for morphological anomalies. A total of 200 spermatozoa per sample were classified according to their morphology; such as normal and abnormal heads, middle piece, and tail. The sum of the abnormal sperm was expressed as a percentage (16).

Evaluation of sperm chromatin quality and apoptosis 2.4.1. Toluidine blue (TB) test
TB stain is a basic nuclear dye used for metachromatic and orthochromatic staining of chromatin. In fact, this stain is a sensitive structural probe for both sperm DNA structure and sperm chromatin packaging, because the test measures the accessibility of the sperm chromatin DNA phosphate residues for dye molecules, which is dependent on both the protein condition and DNA integrity (17,18). In a study under an optical microscope using a magnification of 100 oculars, the quality of the chromatin of the spermatozoa was determined as a function of the metachromatic coloration of the heads of sperm with the following scores: score = 0 light blue (good chromatin); score 1 = dark blue (light abnormal chromatin); and score 2 = purple and violet (severe chromatin anomaly) (19). The total sperms with scores 1 and 2 were considered TB+ or abnormal chromatin while sperms scoring 0 as TB-or sperm with normal chromatin.

Aniline blue (AB) staining
AB staining was performed as previously described by Hofmann and Hilscher (20). AB staining is a kind of cytochemical test for detection of residual histones and therefore indirectly the presence of lower amounts of protamines in the sperm nucleus. (21).  were place green.

Ethical considerations
All animal procedures were performed and approved in accordance with the guide for

Statistical analysis
Statistical analysis was performed using SPSS v. 22 (IBM, 2013). The results were presented as mean ± SD. A P-value < 0.05 was considered significant.          Hence, it seems alcohol can reduce sperm motility by decreasing sperm normal morphology rate. In another study, the epididymal sperm motility was shown to decrease in the ethanolconsuming group (29). Similarly, in the current study, alcohol intake decreased sperm normal morphology and progressive motility. Considering that this increase in motility was not significant in the ethanol 10% group compared to the control group, it seems that the antioxidant system of the mouse body was able to prevent the effect of ethanol 10% on sperm motility, but in the ethanol 20% group decrease in motility was significant, which may indicate the threshold dependent manner effect of ethanol on sperm motility.

Discussion
OS is detrimental to cell function like sperm motility and many hypotheses have been proposed to explain the association between OS and decreased sperm motility (30,31). and epididymides in rats (14).
In the process of spermiogenesis, the nuclei of the spermatids are conditioned, and in this phenomenon, the histones are replaced by nuclear proteins specific to the testes, called Zhu and colleagues showed that in alcoholic men, the rate of apoptosis had increased in germ cells (41). This finding had been assessed based on the fragmentation of sperm DNA by using the TUNEL assay (39). The TUNEL assay is also used to detect DNA strand breakage which is the major sign of sperm apoptosis (42). Our findings in the current study demonstrated that significant differences were found for chromatin damages in ethanol-consumer groups. One possible mechanism for increasing sperm nuclear DNA damage in alcohol-consuming mice is OS.
ROS is the major source of DNA damage, strand breakage, and a variety of alterations in the nucleotides (43).
Although in the present study we did not

Conclusion
The findings of this study showed that sperm forward progressive motility, normal morphology rate, and viability decreased significantly in ethanol-treated groups; also, the rates of spermatozoa with abnormal DNA structure and DNA fragmentation increased significantly in the ethanol-taking groups than the control group. While a cotreatment with vit. E could prevent some of these adverse effects, it can be concluded that vit. E is able to counteract the ethanol-induced impairment of sperm parameters, abnormal DNA structure, packaging, and fragmentation.