Abstract
Objective(s):
Nicotine as a toxic substance leads to impairment of the reproductive system function. The aim of this study was to evaluate effects of melatonin on testicular alterations, sperm nuclear integrity, and epididymal sperm parameters in mice treated with nicotine.
Materials and Methods:
Male mice were divided into four groups. Group A received the vehicle, group B received nicotine 0.1 mg/100 g BW, group C received melatonin 10 mg/kg, group D received nicotine plus melatonin. Evaluations were made by histology and Johnson’s score for study of spermatogenesis, immunostaining for study of male germ cells apoptosis, sperm chromatin dispersion (SCD) test for assaying sperm chromatin integrity, enzyme-linked immunosorbent assay (ELISA) for assessment of serum levels of testosterone and luteinizing hormone (LH), and sperm parameters including morphology, motility, and count.
Results:
Nicotine caused a significant decrease in spermatogenesis quantity and Johnson’s score, sperm parameters, and sex hormones. Melatonin in group D, increased sperm chromatin integrity, improved spermatogenesis, Johnson’s score, and sperm parameters (P<.01) and reduced apoptosis (P <0.01) in comparison with the nicotine group. Melatonin significantly increased testosterone and halo sperms. However, its effect on the LH level was insignificant. The serum testosterone and LH levels were negatively correlated with the DNA fragmentation index (DFI) (r= -0.86, P<0.001) and (r= -0.78, P<0.001), respectively.
Conclusion:
this study showed administration of melatonin in nicotine-treated mice increases both quality and quantity of spermatogenesis and integrity of sperm’s chromatin through reducing apoptosis and modifying the testosterone level.
Keywords: Apoptosis, Chromatin, Melatonin, Nicotine, Sperm, Testis
Introduction
Infertility is an important problem and has adverse effects on various psychological, social, personal, and economic aspects. About 50% of infertility is due to male factors (1). There is a significant relationship between smoking and infertility in men (2).
Nicotine is the most toxic compound present in the cigarette smoke (3). It has adverse effects on the reproductive system in both women (4, 5) and men (6, 7). The use of nicotine reduces gametogenesis and steroidogenesis and inhibits secretion of gonadotropic hormones (8).
Different studies demonstrated that nicotine consumption reduces testosterone release (6, 9, 10), estradiol (4, 5), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) (8). Nicotine reduces fertility in men by affecting the number, movement, survival, and reduction of normal morphology of sperms (11, 12). It also reduces the weight of the testes, body, and libido (10, 11). In addition, through changes in the pituitary-hypothalamic axis it stimulates the release of cortisol, vasopressin, and oxytocin, therefore it increases oxidative stress (11, 13). Meanwhile, it has been shown that nicotine usage in both women and men is associated with an increase in apoptosis in the reproductive system (14-17).
Highly antioxidant compounds may be effective in reducing the effects of nicotine. Melatonin is considered as the main product of pineal gland with a powerful antioxidant activity (18). It can reduce the toxicity of drugs (18). Melatonin has a role in some physiological function such as sleeping, waking up, reproduction, aging, immune response, and tumor growth by itself (19). Melatonin in men is absorbed by the testicles, regulates the activity of the testis and also synthesis by the testes (20). Melatonin through binding receptors can regulate secretion of testosterone and increase the response of the Sertoli cells to FSH during the development of the testicle. It also regulates cell growth, mitosis, and secretion of some types of cells in the testes (20).
Protective effects of melatonin are shown in testis damaged by chemotherapy (21), diabetes (22), hyperlipidemia, and testicular torsion (20). However, there are some reports about effects of nicotine on the male reproductive system but little is known about the effect of nicotine plus melatonin on the testis. The purpose of this study was to investigate effects of exogenous melatonin on spermatogenesis, sperm parameters, and nuclear sperm integrity in adult mice treated with nicotine.
Materials and Methods
Animals
A total of 32 adult NMRI male mice aged 10–12 weeks and weighing 40–45 g were obtained from the Razi Institute, Tehran, Iran and divided into four different groups (8 per group). The animals were housed in small groups under standard lighting conditions (12 hr light-dark cycles) at 25±2 °C with free access to water and food. They were allowed to adapt for at least 1 week in the animal room before they were subjected to treatment. Animals were maintained and handled according to the protocols approved by the Animal Care and Use Committee of Guilan University of Medical Sciences. Animals were divided randomly into four groups:
Group A: Mice received normal saline intraperitoneally once daily for 30 days. Group B: Mice received 0.1 mg/100 g body weight nicotine (Sigma USA) intraperitoneally once daily for 30 days. Nicotine was diluted with normal saline (9,15). Group C: Mice received 10 mg/kg body weight, melatonin (Sigma USA) intraperitoneally once daily for 30 days. Group D: Mice received 0.1 mg/100 g body weight nicotine plus 10 mg/kg melatonin once daily for 30 days.
The dose of 0.1 mg/100g BW of nicotine, is equivalent to the human serum levels of nicotine in those who smoke 20 cigarettes per day (11, 15). Animals in all groups were killed by cervical dislocation and dissected on the 31st day. Testes and epididymis were removed from the mice. The right testis was fixed in Bouin’s solution, dehydrated, and embedded in paraffin. Then, 5 μm sections were prepared and were stained with Haematoxylin and Eosin for assessment of quality and quantity of spermatogenesis. For detection of apoptosis immunohistochemical assessments by TUNEL was performed.
Spermatogenesis assessment
To evaluate spermatogenesis, an optical microscope was used. Using Johnson’s scoring method, the quality and maturation of spermatogenesis in seminiferous tubules were scored from 1 to 10 (21). Also, the number of germ cells including spermatogonia, primary spermatocyte leptotene and pachytene cells, and elongated and round spermatids in the transverse section of 20 seminiferous tubules were counted. It should be noted that the tubules that were in stages VII & VIII were counted. Counting was done in the area of 1 mm2 in each animal, three slides were evaluated.
Assessment of apoptosis
To evaluate apoptosis in germ cells, the TUNEL method was used. The principles of work are based on the guideline contained in the kit (Roche, Germany). In each animal, the number of apoptotic cells in the transverse section of 20 seminiferous tubules was counted and averaged.
Hormone measurement
Blood samples were collected from inferior vena cava immediately after sacrificing the mice. The serum was separated and stored at –80 °C. Testosterone and LH levels were measured using the ELISA kit (Monobind, USA).
Epididymal sperm parameters
For evaluation of the sperm parameters, the tail of epididymis was used, it was moved to a petri dish containing Ham’s F10 media that was warmed to 37 °C previously. The Epididymis was cut into small pieces using a scalpel and then put in an incubator maintained at 37 °C and 5 % CO2 for half an hour.
Following pipetting, 10 µl of the solution was placed on a microscopic slide to evaluate motility. In each animal at least 5 microscopic fields with 400× magnification were studied and percent of motile sperms was specified. Motile sperms were classified into rapid and slow progressive and nonprogressive sperms.
Using a Neubauer slide and 10 µl sperm solution, the number of sperms in 5 cells was counted and expressed as per milliliter. For the study of sperm’s morphology, 10 µl sperm suspension was smeared and dried in air and fixed in 96% ethanol and then stained with Haematoxylin-Eosin. Using a light microscope morphology was evaluated and abnormalities in head and tail was expressed as percentage. In each animal 200 cells were observed with 1000× magnification.
Sperm nuclear DNA integrity
Using sperm chromatin dispersion test (SCD), the integrity of sperm chromatin and fragmentation of sperm’s DNA was measured. At first, the processed sperm solution was diluted with PBS (1:3). Then 30 µl sperm solution and 70 µl low melted agarose were mixed at 37 °C. 10 µl of the above mixed solution was put on a slide covered by 0.65% agarose. After that, a lamella was put on the slide and transferred into the refrigerator and left for 4 min. Then the lamella was removed slowly in a horizontal position. Slides were put in HCL 0.12 N for a duration of 7 min and left in the dark. They were then immersed in the acid denaturation solution containing 2-mercaptoethanol l0.8 m, acidic Tris 0.4 m sodium dodecyl sulfate (SDS) 1%, ethylenediaminetetraacetic acid (EDTA) 50 mM, and sodium chloride (NaCl) 2 m in room temperature for 25 min. Then slides were washed in distilled water two times. They were stained with PBS-Wright (1:1) for 10 min. Observations were done with a light microscope (Olympus, Japan) with 400× magnification. In each animal 200 cells were studied. The fragmented cells had no hallo however, the normal cells with normal DNA strands had a big or small pink hallo around their nucleus. To measure the integrity of sperm chromatin, the percentage of total big and small hallo cells was calculated. DNA fragmentation index (DFI) was measured by counting the ratio of halo-free cells to total cells and was expressed as a percentage.
Statistical analysis
For statistical analysis, the SPSS software package was used. The data were analyzed by analysis of variance (ANOVA) and Tukey’s post hoc tests. Pearson correlation coefficients were used for assaying the relationship between variables. P <0.05 was considered statistically significant.
Results
Nicotine in group B caused a significant decrease in germ cell count, spermatogenesis maturation (Johnsen’s score), and germ cell number in comparison with the controls (P<0.001).
However, in all nicotine-treated animals, all types of male germ cells and Sertoli cells could be observed (Tables 1, 2). An insignificant decrease was observed in spermatogonia cells (Table 1).
Table 1.
Effects of nicotine and melatonin on male mouse germ cell count
| Groups | Spermatogonia | Leptotene P.S. | Pachytene P.S | Round spermatid | Elongated spermatid |
|---|---|---|---|---|---|
| A | 11.38±0.53 | 23.37±0.37 | 35.25±0.52 | 121.38±0.9 | 83.87±3.16 |
| B | 9.80±0.36 | 15.00±0.53a | 18.62±0.67a | 95.5±2.29a | 67.5±1.87a |
| C | 11.50±0.56 | 21.5±0.77b | 33.5±09b | 118.50±1.32b | 82.87±1.68b |
| D | 10.15±0.48 | 20.12±0.63ab | 27.37±1.01ab | 109.71±2.27ab | 79.12±2.72b |
Data are expressed as (mean±standard error)A: controls, B: nicotine group, C: melatonin group D: nicotine+ melatonin P.S.: Primary Spermatocytea:significant compared with control (P<0.001)b: significant compared with the nicotine group (P<0.01)
Table 2.
Effects of nicotine and melatonin on male mouse germ cell parameters and sperm chromatin integrity
| Groups | Apoptotic cells (n) | Johnsen’s score | Halo sperm (%) | SDFI (%) |
|---|---|---|---|---|
| A | 5.43±0.45 | 9.53 ±0.06 | 73.27±1.01 | 12.25±0.53b |
| B | 13.87±0.89a | 8.35±0.16a | 55.88±1.27a | 32.25±0.81a |
| C | 7.25±0.53b | 9.30 ±0.06b | 73.62±1.54b | 10.50±0.7b |
| D | 7.78±0.44b | 9.05±0.06ab | 62.5±2.09ab | 20.28±0.81b |
Data are expressed as (mean±standard error)A: controls, B: nicotine group, C: melatonin group D: nicotine+ melatonina: significant in comparison with controln: number (P<0.001)b: significant compared with the nicotine group (P<0.01)Sperm DNA fragmentation index (SDFI)
Melatonin in a dose of 10 mg/kg had no adverse effects on spermatogenesis quality or quantity in comparison with the controls. However, administration of nicotine plus melatonin in group D increased germ cells in stages of leptotene and pachytene primary spermatocytes, round and elongated spermatids when compared with the nicotine only treated group (Table 1) and (Figures 1 and 2).
Figure 1.
Photomicrograph of seminiferous tubules in mice. Bm: basement membrane, Sg: spermatogonia, Ps: pachytene primary spermatocyte, Ls: leptotene primary spermatocyte, Rs: Round spermatid, Es: elongated spermatid, L: lumen, Le: Leydig cell. S: Sertoli cell. The arrow shows the thickness of germinal epithelium (GE). Haematoxylin-Eosin. 400×
Figure 2.
Histology of the testis in mice. A: controls, B: nicotine group, C: melatonin group, D: nicotine+ melatonin. GE: germinal epithelium, L: lumen, Le: Leydig cells in interstitial tissues. In picture B notice reduced Johnson’s score and germ cells especially elongated spermatids
In controls’ seminiferous tubules the apoptotic cells were scattered. Administration of nicotine in doses of 0.1 mg/100 g weight once daily for 30 days significantly increased apoptotic cells (P<0.01). Apoptotic cells were mostly observed in primary spermatocytes and also elongated spermatids. Melatonin in the last group reduced the apoptotic cells in comparison with the nicotine-treated group (Table 2) (Figure 3). Melatonin also increased germ cell count and Johnsen’s score in comparison with the nicotine group (P<0.01) (Table 2) (Figure 3).
Figure 3.
Immunostaining of seminiferous tubules. A: controls, B: nicotine group, C: melatonin group, D: nicotine+ melatonin group. White arrows show brilliant apoptotic germ cells. TUNEL. 400×
Nicotine reduced sperm count, motility and normal morphology in epididymal sperms when compared with controls (P<0.001). It also reduced halo cells (P<0.001). DNA fragmentation index (DFI) was increased in the nicotine group (P<0.001). Administration of melatonin in the last group increased the abovementioned parameters in epididymal sperms compared with the nicotine-treated group (P<0.01) (Tables 2, 3) and (Figure 4).
Table 3.
Effects of nicotine and melatonin on epididymis sperm parameters
| Groups | Count (×10 6/ml) | Rapid progressive motility (%) | Slow progressive Motility (%) | No progressive motility (%) | Total motility (%) | Abnormal morphology (%) |
|---|---|---|---|---|---|---|
| A | 32.52±1.46 | 10.1±0.17 | 38.4±2.16 | 15.11±0.27 | 63.97±1.24 | 27.50±1.16 |
| B | 23.43±0.75a | 2.4±0.2a | 30.2±2.83 | 30.41±0.4a | 41.07±1.37a | 45.50±0.85a |
| C | 33.08±0.88b | 12.5±0.13 | 44.2±2.23 | 10.5±2.48 | 67.62±1.90b | 25.87±0.5b |
| D | 29.82±0.80b | 8.6±0.06 | 35.4±2.58 | 18.61±2.9ab | 63.75±2.68b | 29.62±1.03b |
Data are expressed as (mean±standard error)A: controls, B: nicotine group, C: melatonin group, D: nicotine+ melatoninSDFI: Sperm DNA Fragmentation indexa: significant in comparison with controls (P <0.001)b: significant compared with the nicotine group (P <0.01)
Figure 4.
light photomicrograph from halo sperms in control mice. 1: big halo spermatozoa, 2: small halo spermatozoa, 3: degraded spermatozoa. Sperm chromatin dispersion test. 400×
A significant decrease in serum LH and testosterone levels was observed in group B in comparison with controls (P <0.001). Administration of nicotine plus melatonin significantly increased the testosterone level compared with the nicotine-treated group (P <0.001). The amount of LH in serum in the last group was a little higher than in the nicotine-treated group but it was insignificant (P >0.05) (Figure 5).
Figure 5.
Effects of nicotine and melatonin on serum testosterone and LH levels. Data are expressed as (mean±standard error). A: controls, B: nicotine group, C: melatonin group, D: nicotine+ melatonin. a: significant compared with controls (P <0.001). b: significant compared with the nicotine group (P <0.001)
The serum testosterone level was negatively correlated with the DFI (r= -0.86, P <0.001). There was a negative correlation between the LH level and DFI (r= -0.78, P <0.001). Abnormal sperm morphology showed a positive correlation with DFI (r=0.85, P <0.01). Male germ cell apoptosis positively correlated with DFI (r=0.78, P <0.01). Sperm motility showed a negative correlation with DFI (r=- 0.79 P <0.001).
Discussion
The present study showed administration of nicotine in a dose of 0.1 mg/100 g BW for 30 days in adult male mice caused adverse changes in spermatogenesis, apoptosis, sperm parameters, and sperm chromatin integrity. This effect is likely due to alterations in pituitary gonadotrophins essential for initiating and completing spermatogenesis and steroidogenesis in the testis. Because nicotine can suppress central nervous system, which can inhibit the neural stimulus essential for the release of gonadotrophins from hypophysis (23). Studies have implicated nicotine in infertility due to affecting sperm function by decreasing the sperm count motility, morphology, viability, and testicular weight (24, 25).
Spermatogenesis is a highly regulated process and is controlled by hormones especially testosterone and gonadotropins FSH and LH (8, 26). Our ELISA assay revealed a significant decrease in serum testosterone and LH levels in the nicotine group. Testosterone is a key hormone for the spermatogenesis process and is required for beginning and maintenance of spermatogenesis. LH is secreted from hypophysis and affects the Leydig cells and stimulates them for secretion of testosterone. Therefore it seems reduced LH causes reduced testosterone and our results confirm it.
Nicotine and cotinine are both 3α-hydroxysteroid dehydrogenase inhibitors, which is an important enzyme in the metabolism of testosterone and dihydrotestosterone and plays a role in changing the function of androgens in testosterone-dependent tissues such as prostate (6). In this study, contrary to the fact that most germ cells were reduced after nicotine use, spermatogonia cells did not decrease significantly. Perhaps this difference is due to the presence of different receptors on the various types of germ cells in the testes (27).
Apoptosis is a programmed physiological death that can be created in different types of cells during different periods of life. Increased apoptosis in this study may be due to nicotine’s own toxic agent (24, 25) or due to the reduction of testosterone and LH, which our study confirms. It may also be due to the higher levels of oxidants in nicotine-treated groups. In this regard, it has been shown that nicotine reduces the testicular antioxidant level and increases testicular lipid peroxidation (11).
In the present study, we performed the SCD test for evaluating sperm chromatin integrity. The traditional evaluation of sperm to study the fertility status of men is not enough and the evaluation of the sperm DNA yields more information about fertility (26). High levels of sperm DNA damage are associated with reduced fertility (26), and nowadays oxidative stress is considered as a strong factor for single- and double-strand deoxyribonucleic acid (DNA) breaks (27) effect (28).
Our study showed nicotine increased both abnormal sperms and fragmentation of sperm’s DNA. Condorelli et al. in an in vitro study, showed that nicotine, depending on the dose and time, reduced sperm motility and live sperm, and increased sperm percentage by decreasing the mitochondrial membrane potential and the integrity of the chromatin DNA of the sperm (29).
Our study showed a negative correlation between testosterone and LH sex hormones with SCD. In this regard, a previous study on humans, has shown that there is a negative relationship between testosterone and estradiol hormones with sperm’s DFI in fertile men (30). In confirmation of our findings regarding the existence of a negative link between motility and DFI, some studies have shown a significant negative relationship between sperm motility and sperm DNA damage (31, 32).
This study showed melatonin in the last group improved both spermatogenesis and sperm parameters. Previous studies have shown that melatonin reduces adverse effects of nicotine on the ovary and uterus (4, 5). It also increases spermatogenesis and sperm parameters in mice treated with chemotherapy (21) and acetylsalicylic acid (33) and in diabetic rats (34). It also modifies spermatogenesis in hyperlipidemic animals and following testicular torsion (20).
Melatonin is a hormone synthesized mainly in the pineal gland and also in the other organs such as retina and lacrimal glands (35). The concentration of melatonin in the testicular tissue is similar to that of other tissues in the body (26). Studies have shown that melatonin has high antioxidant properties and the potential to trap free radicals (26). Sperm exposure to melatonin in vitro results in better semen quality (36). Therefore these findings show the beneficial effects of melatonin on sperms.
The better spermatogenesis status in the last group may be due to anti-apoptotic and antioxidant effects of melatonin (4, 22) and our study confirmed it. Melatonin also affects the hypothalamic–pituitary–gonadal (HPG) axis and modifies the secretion of sex hormones such as estrogen, testosterone, FSH, and LH (19). In confirmation of these, in our study, melatonin also significantly increased testosterone and partly the LH hormone and improved DFI in the last group compared with the nicotine group. This is likely due to the direct effect of melatonin on Leydig cells and in this regard, the presence of melatonin receptors on the rat Leydig cells has been shown (37). Melatonin increased the integrity of sperm DNA in the nicotine-treated group. Casao et al. showed that there is a relation between melatonin and testosterone hormones and antioxidant enzymes in ram seminal plasma (38). Therefore these studies confirm that melatonin has an effect on sperm’s DNA fragmentation index.
The exact mechanism of the effect of melatonin-nicotine on the quality of spermatogenesis is not clear and requires more studies.
Conclusion
This study showed administration of melatonin in nicotine-treated mice increases both quality and quantity of spermatogenesis and integrity of sperm chromatin through reducing apoptosis and modifying the testosterone level.
Ethical Statement
Animals were maintained and handled according to the protocols approved by the Animal Care and Use Committee of Guilan University of Medical Sciences, Rasht, Iran.
Acknowledgment
This work was supported financially by Deputy of Research and Technology, Guilan University of Medical Sciences, Rasht, Iran. There are no conflicts of interest to declare.
References
- 1.Harlev A, Agarwal A, Gunes SO, Shetty A, du Plessis SS. Smoking and male Infertility: an evidence-based review. World J Mens Health. 2015;33:143–160. doi: 10.5534/wjmh.2015.33.3.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Lagunov A, Anzar M, Sadeu JC, Khan MI, Bruin JE, Woynillowicz AK, et al. Effect of in utero and lactational nicotine exposure on the male reproductive tract in peripubertal and adult rats. Reprod Toxicol. 2011;31:418–423. doi: 10.1016/j.reprotox.2010.12.004. [DOI] [PubMed] [Google Scholar]
- 3.Mosbah R, Yousef MI, Mantovani A. Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: the protective effects of green tea extract. Exp Toxicol Pathol. 2015;67:253–259. doi: 10.1016/j.etp.2015.01.001. [DOI] [PubMed] [Google Scholar]
- 4.Saadat SN, Mohammadghasemi F, Jahromi SK, Homafar MA, Haghiri M. Melatonin protects uterus and oviduct exposed to nicotine in mice. Interdiscip Toxicol. 2014;7:41–46. doi: 10.2478/intox-2014-0007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mohammadghasemi F, Jahromi SK, Hajizadeh H, Homafar MA, Saadat N. The protective effects of exogenous melatonin on nicotine-induced changes in mouse ovarian follicles. J Reprod Infertil. 2012;13:143–150. [PMC free article] [PubMed] [Google Scholar]
- 6.Carvalho CA, Favaro WJ, Padovani CR, Cagnon VH. Morphometric and ultrastructure features of the ventral prostate of rats (Rattus norvegicus) submitted to long-term nicotine treatment. Andrologia. 2006;38:142–151. doi: 10.1111/j.1439-0272.2006.00728.x. [DOI] [PubMed] [Google Scholar]
- 7.Gu Y, Xu W, Nie D, Zhang D, Dai J, Zhao X, et al. Nicotine induces Nm e2-mediated apoptosis in mouse testes. Biochem Biophys Res Commun. 2016;472:573–579. doi: 10.1016/j.bbrc.2016.03.044. [DOI] [PubMed] [Google Scholar]
- 8.Jana K, Samanta PK, De DK. Nicotine diminishes testicular gametogenesis, steroidogenesis, and steroidogenic acute regulatory protein expression in adult albino rats: possible influence on pituitary gonadotropins and alteration of testicular antioxidant status. Toxicol Sci. 2010;116:647–659. doi: 10.1093/toxsci/kfq149. [DOI] [PubMed] [Google Scholar]
- 9.Oyeyipo IP, Raji Y, Bolarinwa AF. Nicotine alters male reproductive hormones in male albino rats: The role of cessation. J Hum Reprod Sci. 2013;6:40–44. doi: 10.4103/0974-1208.112380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Abd El-Aziz GS, El-Fark MO, Hamdy RM. Protective effect of Eruca sativa seed oil against oral nicotine induced testicular damage in rats. Tissue Cell. 2016;48:340–348. doi: 10.1016/j.tice.2016.05.006. [DOI] [PubMed] [Google Scholar]
- 11.Oyeyipo IP, Raji Y, Bolarinwa AF. Antioxidant profile changes in reproductive tissues of rats treated with nicotine. J Hum Reprod Sci. 2014;7:41–46. doi: 10.4103/0974-1208.130823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Reddy A, Sood A, Rust PF, Busby JE, Varn E, Mathur RS, et al. The effect of nicotine on in vitro sperm motion characteristics. J Assist Reprod Genet. 1995;12:217–223. doi: 10.1007/BF02211802. [DOI] [PubMed] [Google Scholar]
- 13.Nesseim WH, Haroun HS, Mostafa E, Youakim MF, Mostafa T. Effect of nicotine on spermatogenesis in adult albino rats. Andrologia. 2011;43:398–404. doi: 10.1111/j.1439-0272.2010.01086.x. [DOI] [PubMed] [Google Scholar]
- 14.Kim KH, Joo KJ, Park HJ, Kwon CH, Jang MH, Kim CJ. Nicotine induces apoptosis in TM3 mouse Leydig cells. Fertil Steril. 2005;83(Suppl1):1093–1099. doi: 10.1016/j.fertnstert.2004.12.013. [DOI] [PubMed] [Google Scholar]
- 15.Nie D, Zhang D, Dai J, Zhang M, Zhao X, Xu W, et al. Nicotine Induced murine spermatozoa apoptosis via up-regulation of deubiquitinated RIP1 by Trim27 promoter hypomethylation. biol Reprod. 2016;94:31. doi: 10.1095/biolreprod.115.131656. [DOI] [PubMed] [Google Scholar]
- 16.Patil SR, Ravindra Patil SR, Londonkar R, Patil SB. Nicotine induced ovarian and uterine changes in albino mice. Indian J Physiol Pharmacol. 1998;42:503–508. [PubMed] [Google Scholar]
- 17.Petrik JJ, Gerstein HC, Cesta CE, Kellenberger LD, Alfaidy N, Holloway AC. Effects of rosiglitazone on ovarian function and fertility in animals with reduced fertility following fetal and neonatal exposure to nicotine. Endocrine. 2009;36:281–290. doi: 10.1007/s12020-009-9229-4. [DOI] [PubMed] [Google Scholar]
- 18.Reiter RJ. Oxidative damage in the central nervous system: protection by melatonin. Prog Neurobiol. 1998;56:359–384. doi: 10.1016/s0301-0082(98)00052-5. [DOI] [PubMed] [Google Scholar]
- 19.Sanchez-Hidalgo M, de la Lastra CA, Carrascosa-Salmoral MP, Naranjo MC, Gomez-Corvera A, Caballero B, et al. Age-related changes in melatonin synthesis in rat extrapineal tissues. Exp Gerontol. 2009;44:328–334. doi: 10.1016/j.exger.2009.02.002. [DOI] [PubMed] [Google Scholar]
- 20.Frungieri MB, Calandra RS, Rossi SP. Local Actions of Melatonin in Somatic Cells of the Testis. Int J Mol Sci. 2017:18. doi: 10.3390/ijms18061170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Mohammad Ghasemi F, Faghani M, Khajeh Jahromi S, Bahadori M, Nasiri E, Hemadi M. Effect of melatonin on proliferative activity and apoptosis in spermatogenic cells in mouse under chemotherapy. J Reprod Contraception 2010. 21:79–94. [Google Scholar]
- 22.Armagan A, Uz E, Yilmaz HR, Soyupek S, Oksay T, Ozcelik N. Effects of melatonin on lipid peroxidation and antioxidant enzymes in streptozotocin-induced diabetic rat testis. Asian J Androl. 2006;8:595–600. doi: 10.1111/j.1745-7262.2006.00177.x. [DOI] [PubMed] [Google Scholar]
- 23.Aydos K, Güven M, Can B, Ergün A. Nicotine toxicity to the ultrastructure of the testis in rats. BJU Int. 2001;88:622–626. doi: 10.1046/j.1464-4096.2001.02384.x. [DOI] [PubMed] [Google Scholar]
- 24.Oyeyipo IP, Raji Y, Emikpe BO, Bolarinwa AF. Effects of oral administration of nicotine on organ weight, serum testosterone level and testicular histology in adult male rats. Niger J Physiol Sci. 2010;25:81–86. [PubMed] [Google Scholar]
- 25.Oyeyipo IP, Raji Y, Emikpe BO, Bolarinwa AF. Effects of nicotine on sperm characteristics and fertility profile in adult male rats: a possible role of cessation. J Reprod Infertil. 2011;12:201–207. [PMC free article] [PubMed] [Google Scholar]
- 26.Malm G, Haugen TB, Rylander L, Giwercman A. Seasonal fluctuation in the secretion of the antioxidant melatonin is not associated with alterations in sperm DNA damage. Asian J Androl. 2017;19:52–56. doi: 10.4103/1008-682X.186870. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Aitken RJ, Koopman P, Lewis SE. Seeds of concern. Nature. 2004;432:48–52. doi: 10.1038/432048a. [DOI] [PubMed] [Google Scholar]
- 28.Sakkas D, Urner F, Bizzaro D, Manicardi G, Bianchi PG, Shoukir Y, et al. Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development. Hum Reprod. 1998;13(Suppl4):11–19. doi: 10.1093/humrep/13.suppl_4.11. [DOI] [PubMed] [Google Scholar]
- 29.Condorelli RA, La Vignera S, Giacone F, Iacoviello L, Vicari E, Mongioi L, et al. In vitro effects of nicotine on sperm motility and bio-functional flow cytometry sperm parameters. Int J Immunopathol Pharmacol. 2013;26:739–746. doi: 10.1177/039463201302600317. [DOI] [PubMed] [Google Scholar]
- 30.Richthoff J, Spano M, Giwercman YL, Frohm B, Jepson K, Malm J, et al. The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA) Hum Reprod. 2002;17:3162–3169. doi: 10.1093/humrep/17.12.3162. [DOI] [PubMed] [Google Scholar]
- 31.Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod. 2003;18:1023–1028. doi: 10.1093/humrep/deg228. [DOI] [PubMed] [Google Scholar]
- 32.Giwercman A, Richthoff J, Hjollund H, Bonde JP, Jepson K, Frohm B, et al. Correlation between sperm motility and sperm chromatin structure assay parameters. Fertil Steril. 2003;80:1404–1412. doi: 10.1016/s0015-0282(03)02212-x. [DOI] [PubMed] [Google Scholar]
- 33.Hedayati Emami N, Mahmoudi lafout F, Mohammadghasemi F. Administration of melatonin protects against acetylsalicylic acid-induced impairment of male reproductive function in mice. Iran J Basic Med Sci. 2018;21:124–129. doi: 10.22038/IJBMS.2017.23833.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.da Costa CF, Gobbo MG, Taboga SR, Pinto-Fochi ME, Goes RM. Melatonin intake since weaning ameliorates steroidogenic function and sperm motility of streptozotocin-induced diabetic rats. Andrology. 2016;4:526–541. doi: 10.1111/andr.12158. [DOI] [PubMed] [Google Scholar]
- 35.Hemadi M, Saki G, Shokri S, Ghasemi FM. Follicular dynamics in neonate vitrified ovarian grafts after host treatment with melatonin. Folia Morphol (Warsz) 2011;70:18–23. [PubMed] [Google Scholar]
- 36.Ortiz A, Espino J, Bejarano I, Lozano GM, Monllor F, Garcia JF, et al. High endogenous melatonin concentrations enhance sperm quality and short-term in vitro exposure to melatonin improves aspects of sperm motility. J Pineal Res. 2011;50:132–139. doi: 10.1111/j.1600-079X.2010.00822.x. [DOI] [PubMed] [Google Scholar]
- 37.Balik A, Kretschmannova K, Mazna P, Svobodova I, Zemkova H. Melatonin action in neonatal gonadotrophs. Physiol Res. 2004;53(Suppl 1):S153–166. [PubMed] [Google Scholar]
- 38.Casao A, Cebrian I, Asumpcao ME, Perez-Pe R, Abecia JA, Forcada F, et al. Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes. Reprod Biol Endocrinol. 2010;8:59. doi: 10.1186/1477-7827-8-59. [DOI] [PMC free article] [PubMed] [Google Scholar]