Effects of ethanol consumption on chromatin condensation and DNA integrity of epididymal spermatozoa in rat

Abstract

Alcohol abuse is considered as one of the problems associated with poor semen production and sperm quality. Both acute and chronic alcohol consumption may affect spermatozoal chromatin disorders through apoptosis. Therefore, for the first time, this experimental study was performed to evaluate the effect of ethanol consumption on sperm parameters and chromatin integrity of spermatozoa aspirated from cauda epididymis of rats. Twenty adult Wistar rats were divided into ethanol consumption and control groups. Access to ethanol and water was provided ad libitum for experimental and control animals, respectively. The cauda epididymal spermatozoa were aspirated for analysis of sperm parameters and sperm chromatin integrity with aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. Sperm progressive and nonprogressive motility of ethanol-consuming rats were significantly decreased compared with control animals (P < .05). In addition, the rates of AB-reacted spermatozoa were similar in both groups (P > .05). However, with regard to CMA3, AO, and TB stainings, there was a significant increase in ethanol group when compared with the controls (P < .05). The majority of TB+ and AO+ spermatozoa were higher than “cut-off” value in ethanol group, whereas the mean rates of CMA3+ spermatozoa was below the “cut-off” value in both groups. The results showed that ethanol consumption disturbs sperm motility, nuclear maturity and DNA integrity of spermatozoa in rat. Therefore, ethanol abuse results in the production of spermatozoa with less condensed chromatin, and this may be one possible cause of infertility following ethanol consumption.

Introduction

Infertility is a major problem in up to 15% of the sexually active population and male factor is responsible in 50% of these cases (World Health Organization, 1999). Recently, the substances in the environment that can disturb male fertility have been increased. Ethanol is among the most widely abused drug, which can suppress reproductive function and sexual behavior in laboratory animals and humans (Abel, 1980, Fadem, 1993). The lack of sexual desire in long-term alcohol users has been reported from 31 to 58% (Gümüş et al., 1998, Jensen, 1984, Whalley, 1978). Whalley (1978) reported that, about 54% of hospitalized alcoholic men and 24% of healthy controls had erectile dysfunction. Alcohol is toxic for testes and causes fertility disturbances through low sperm count and motility in men (Maneesh et al., 2006). In addition, Van Thiel et al., 1980, Van Thiel et al., 1975 showed that chronic ethanol exposure decreased plasma testosterone level and caused testicular atrophy. Irregular diameter of the seminiferous tubules and high amount of death cells in the lumen of alcoholic men were also noticed by Martinez et al. (2009).

Sperm nuclear DNA in mammalians is organized around protamine molecules during testicular spermiogenesis. In this phase, sperm-specific histones are replaced by protamines, which are rich in cysteine and other basic amino acids. During epididymal transit, the cysteine-thiol group of protamine molecules are oxidized to disulphide bonds (S–S), which are necessary for stability of sperm chromatin (Poccia, 1986, Said et al., 1999). In the cases of reduction in the number of S–S bonds between protamine molecules, the chromatin will be more susceptible to denaturation. In rats, spermatozoa contain approximately 84% of total SH and S–S groups in the epididymal caput as thiols. This is decreased to 14% in spermatozoa obtained from the cauda epididymis (Said et al., 1999). This difference indicates that during transit between the two epididymal regions, about 1.5 billion S–S bonds are formed per spermatozoa. Therefore, spermatozoa become highly resistant to a variety of agents such as acids, proteases, DNase and detergents such as sodium dodecyl sulfate after nuclear chromatin condensation (Mahi and Yanagimachi, 1975). Evenson et al. (1999) reported that sperm chromatin condensation is a complex process, which is directly related to the capacity of sperm to fertilize an oocyte. Indeed, sperm DNA damages, such as DNA fragmentation, abnormal chromatin packaging, and protamine deficiency, have been correlated with the reduced ability of spermatozoa to fertilize oocyte in the context of assisted reproduction techniques and normal fertility (Ahmadi and Ng, 1999, Cho et al., 2003, Filatov et al., 1999, Lopes et al., 1998, Sakkas et al., 1996).

Regarding to alcohol consumption, although there is a significant association between alcohol intake and increasing frequency of sperm nuclear aneuplody (Robbins et al., 1997), but little is known about the impact of ethanol toxicity on sperm chromatin and DNA integrity. Zhu et al. (2000) reported that ethanol exposure enhanced the testicular germ cells apoptosis and the increased expression of Fas ligand was remarkable in rats, which were fed ethanol chronically. Moreover, both acute and chronic alcohol exposure can increase production of reactive oxygen species (ROS) (Wu and Cederbaum, 2003). Alcohol metabolism produces the reduced form of nicotinamide adenine dinucleotide (NADH), which enhances activity of the respiratory chain and ROS formation. In addition, one of the by-products of alcohol metabolism, acetaldehyde, interact with proteins and lipids to form ROS. It should be noticed that if levels of ROS rise above the body’s antioxidant defense system, oxidative stress (OS) occurs (Agarwal and Prabakaran, 2005, Goverde et al., 1995).

It is generally accepted that plasma membrane and DNA molecules are the major targets of ROS in sperm and other cells. In addition, there is a positive correlation between the level of ROS and sperm apoptosis (Moustafa et al., 2004). So, in the cases of alcohol consumption, sperm nuclear anomalies and apoptosis may be expected due to the OS.

Alcohol drinking is increasing and it is considered as a common social problem. In Australia, one study found that 9% of 14–19 years old participants consumed five (females) or seven (males) drinks or more at least 1 day per week (Hargreaves et al., 2009). In addition, if the ethanol consumption is determined to affect male reproduction through DNA damaging, men who wish to be fertile, should be specially warned of this matter. According to our knowledge, there is no report on effects of ethanol on sperm chromatin condensation using cytochemical assays. Therefore, the aim of this study was to assess the possible detrimental effects of ethanol consumption on sperm DNA integrity and chromatin quality in rat as an experimental model.