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Official websites use. Share sensitive information only on official, secure websites. Normal saline was injected in sham group and no medications were used in control group. To evaluate sperm chromatin abnormalities, the aniline blue, toluidine blue and chromomycine A3 were used. For sperm morphology, Papanicolaou staining was done. There was a significant increase in rate of aniline blue, toluidine blue, and chromomycine A3 positive spermatozoa in high dosage group. In a similar manner, there was an increase in rates of acridine orange, TUNEL and sperm chromatin dispersion positive sperm cells in high dosage group with respect to others. In recent years, methamphetamine MA has been attractive and road drug in several countries, because of its quite easy manufacture and low price versus to another drugs 1. MA is an illegal psycho stimulant drug profitable to amphetamine type 2. MA is a strongly addictive drug with a high probability of addition which is absorbed slowly for a long period of time, consequently for hr 3. It is usually used by young and teenagers who are in the age of reproduction 4. In fact, young population experience MA for fun and improvement of sexual function at first times 5. Earlier, many studies proved negative effects of morphine and cocaine, but today consumption of synthetic drugs including amphetamines or MA is increased in developed and developing countries 6 - 8. The exact mechanism by which MA leads to male reproductive system dysfunctions is not completely understood. There are many studies indicating the deleterious effects of MA on reproductive organs 9. Experimental studies on rodents suggest some mechanisms of MA action on male fertility potential including altered hormonal profiles, oxidative stress, DNA damage of spermatozoa, and abnormal spermatogenesis 10 , It is also demonstrated that MA adversely affects on seminiferous epithelium including degeneration and apoptosis of germ cells It is also suggested that MA affects male reproductive function at multiple levels due to its effects on the endocrine control of spermatogenesis 13 , Recently, it has been reported that MA decreases normal sperm morphology and count, as well as increase apoptotic cells in seminiferous tubules 15 , In a study conducted by Zuloaga et al , histopathological and histomorphometric alterations in seminiferous tubules have been reported in MA-treated animals Evaluation of sperm nuclear chromatin is a noticeable approach for male fertility investigations. During spermatogenesis, sperm chromatin is compacted more and more due to histones replacement at first by testis-specific nuclear proteins, then by transitional proteins and finally by protamines Disulphide bonds of protamine molecules are crucial for sperm nuclear compaction and stabilization. It is believed that this kind of nuclear compaction protects sperm genome from damages including oxidative stress, elevated temperature and acid-induced DNA denaturation Oxidative stress OS is considered as an important cause of male infertility leading to an increase in sperm DNA fragmentation. Imbalance between reactive oxygen species ROS production and semen antioxidant ability results OS It is generally accepted that ROS affects sperm chromatin condensation and also may have harmful effects on sperm motility, morphology and fertilization ability To the best of our knowledge, there are no study that investigated the effects of MA on sperm chromatin condensation and DNA integrity. Therefore, we designed the present study to investigate the effects of different doses of MA on sperm count, motility, and morphology and sperm chromatin integrity in male mouse as an experimental model. They were fed with normal pellet diet and water adlibitum. Normal saline was injected daily in sham group, but, the control mice did not receive any medication. The percentages of progressive fast and slow movements , non-progressive and immotile spermatozoa were calculated For assessment of sperm morphology, the Papanicolaou staining was done. Briefly, the smears were fixed by ethanol-ether for 5 min and then they were stained with PAP staining solutions according to WHO guidelines Different forms of sperm morphology were determined including normal, double head, pin head, amorphous head, coiled mid piece, coiled tail, bent tail, and cytoplasmic droplet Figure 1-A. The AR was assessed by double staining method. The smears were prepared after two times washing rpm, 2 min. The slides were stained with Bismarck brown 0. After washing, smears were dehydrated in ethanol series and rinsed in xylene Red or pink staining of the acrosomal region determined as acrosome-intact spermatozoa. AB staining is a cytochemical assay for detection of remained histones in the process of sperm chromatin remodeling In this staining, the spermatozoa with unstained nucleus are considered as normal and spermatozoa with dark blue nuclei are counted as abnormal ones Figure 1-B. The air-dried smears were fixed in a solution of ethanol-acetone at 4 o C for 30 min. Hydrolysis of smears was performed by HCl 0. Then, TB dye solution 0. Finally, the slides were rinsed in distilled water and dehydrated with ethanol and xylene at room temperature for 3 min Spermatozoa with normal chromatin are seen colorless but sperm cells with mild, medium and sever chromatin abnormality were seen in dark blue, violet and purple respectively Figure 1-C. CMA3 staining was used for indirect assessment of protamine deficiency. The prepared slides were evaluated under fluorescent microscope with nm filter. The bright yellowish spermatozoa were considered as positive CMA3, while others without brightness were considered as negative CMA3 with normal protamine. In the next step, they were immersed in 0. The apoptotic sperm cells were presented as percentage in each sample. This assay is used for detection of sperm DNA damage. Then, coverslip was removed and slide was embedded in 0. Each slide was immersed in lysis solutions 1 and 2 sequentially. The time of lysis solution 1 0. Finally, each slide was rinsed in wright stain for 10 min. The small, medium and large halos around sperm heads were determined in comparison with core width of spermatozoa. The small halo showed high DNA fragmentation and the medium and large ones showed moderate and without DNA fragmentation in spermatozoa respectively 33 Figure 2-C. All protocols were performed according to the national institute of health guide for the care and use of laboratory animals NIH Publications No. For statistical analysis, we used Statistical Package for the Social Sciences, version According to table I , although, the sperm count showed a decreasing trend in medium and high dosage groups when compared to low dosage and control one, but it was not statistically significant. Normal morphology was significantly decreased in medium and high dosage groups compared to other groups. Due to the high prevalence of MA abuse in young populations especially in men who are in reproductive period, the present study was planned to estimate the specific deleterious effects of this central nervous system stimulant as an addictive drug on sperm parameters and chromatin integrity in male mice. The results showed that MA decreases sperm concentration, progressive motility and increases abnormal sperm morphology in a dose-dependent manner. Moreover, sperm chromatin packaging and apoptosis could be influenced by different dose of MA. The effects of MA on sperm chromatin also appear to be dose-depended as similar drugs which had more negative effects in high doses. Our findings were similar to the study by Alavi and colleagues which have demonstrated that frequent consumption of high dosage of MA may reduce the number of sperm in the epididymis tail and has negative effects on reproductive system in male rats Additionally, several experimental studies showed that administration of different dose of MA caused decreasing in the sperm count and normal morphology 35 , On the other hand, Lin and colleagues reported many defects in testicular tissue and spermatogenesis cell lines including the elevation of apoptotic cells and destructive changes in seminiferous tubules in male rats which were treated by MA. In addition, they presented that sperm count and motility were decreased in MA administration animals It is generally accepted that in male fertility evaluations, the standard routine sperm analysis is not enough, because it does not show different forms of sperm chromatin and DNA damages. So, in the present study, we focused on molecular and cytochemically-based assays to determine the effects of MA on sperm chromatin condensation and apoptosis in mouse as a good experimental model. It is shown that there is a positive relationship between sperm chromatin defects and cell death or apoptosis Similar to our study, Yamamoto and colleagues reported that administration of MA reduces the spermatogenesis and increases apoptosis in spermatogonial cell line in rat Additionally, Alavi and his colleagues demonstrated that repeated administration of MA in male rats enhances apoptosis of spermatogonia and primary spermatocytes in seminiferous tubules. In fact, they discussed that decreasing in sperm count and normal morphology and apoptotic cell increasing in seminiferous tubules may be due to the MA toxicity in sperm germ line and testicular tissue Another recent study has reported a reduction of reproductive ability in animals that were administered by MA Also, in a sex hormones assessment, it has been suggested that any alternation in serum testosterone during the MA treatment can affect the spermatogenesis and it has a main role in activation of apoptosis in spermatogonial cells Although in present study we can not report any evidence about the mechanism of harmful characteristics of MA on gonadal hormones and risky effect on spermatogenesis, but it could be mentioned that reduction in sperm chromatin quality was dependent on toxic effects of MA and hormonal profile defects. Recently, it was shown that MA can induce apoptosis in testicular germ cells and epididymal spermatozoa in laboratory animals AB and CMA3 are two main tests for identifying sperm chromatin packaging and protamine deficiency of spermatozoa It is confirmed that the lower amounts of nuclear protamines affect the sperm chromatin packaging and as a result the sperm quality The present study was also the first study to the evaluation of the sperm acrosome in MA administration animals. There was a significant difference between the percentage of spermatozoa with intact acrosomes in experimental and control groups. In conclusion, MA can increase abnormal spermatozoa with inadequate chromatin and DNA integrity in a dose-dependent manner and it is important to aware the young male people from deleterious effects of this drug on reproductive indices. As a library, NLM provides access to scientific literature. Int J Reprod Biomed. Find articles by Mojdeh Sabour. Arezoo Khoradmehr , M. Find articles by Arezoo Khoradmehr. Seyyed Mehdi Kalantar , Ph. Find articles by Seyyed Mehdi Kalantar. Amir Hossein Danafar , M. Find articles by Amir Hossein Danafar. Marjan Omidi , M. Find articles by Marjan Omidi. Iman Halvaei , Ph. Find articles by Iman Halvaei. Ali Nabi , M. Find articles by Ali Nabi. Saeed Ghasemi-Esmailabad , M. Find articles by Saeed Ghasemi-Esmailabad. Ali Reza Talebi , Ph. Find articles by Ali Reza Talebi. PMC Copyright notice. Open in a new tab. Data are shown as median min- max. Effect of different doses of MA on sperm nuclear integrity and apoptosis. Data are shown as median min- max as percentage. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.
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