cigarette smoke contains a mixture of 4000 toxic chemicals , including nicotine , addictive components , carbone monoxide , and several recognized carcinogens and mutagens ( 1 ) . smoking has deleterious effects on cardiovascular , pulmonary physiology and reproductive system ( 1 ) . in women , smoking is associated with infertility , spontaneous abortion , menstrual abnormalities , ectopic pregnancies and early onset of menopause ( 2 , 3 ) . nicotine is a highly toxic substance and it is quickly absorbed through the respiratory tract , mouth mucosa and skin ( 1 ) . quantitatively , the most important metabolite of nicotine in mammalian species and humans is the lactam derivative cotinine . in humans , about 70 to 80% of nicotine is converted to cotinine ( 4 ) . in adult humans , cotinine has been detected in the follicular fluid of women who smoke , demonstrating that nicotine reaches the ovary and developing follicles . treatment of rats with nicotine is associated with a decrease in estrogen dependent parameters , including uterine weight , myometrial and endometrial diameter and thickness ( 1 ) . moreover , nicotine causes a significant decrease in the concentrations of na , k , and cl in the uterine fluid and endometrial cells in situ . nicotine also reduces uterine blood flow by an average of 30 to 49% ( 5 ) . there are different forms of nicotine administration for evaluating its absorption pharmacokinetics such as smoking , nasal spray , gum , inhaler , sublingual tablets , tooth pach , transdermal pach , intravenous and subcutaneous injections , oral capsule , oral solution and enema . in animal models this may be due to the fact that intraperitoneal or subcutaneous routes facilitate the rapid absorbtion of the substance . the oral bioavailability of nicotine is about 20 to 45% ; however , in intraperitoneal or subcutaneous routes it is much more ( about 100% ) . oral bio - availability of nicotine is incomplete because of the hepatic first- pass metabolism ( 4 ) . all of this evidence indicates that nicotine can affect gamete cell function . in vitro studies in luteal cells investigations on nicotine have indicated that nicotine , inhibits the release of gonadotropines from the pituitary gland by affecting the central nervous system ( 6 ) . exposure of the uterus to nicotine causes impaired fertility , altered ovarian steroid hormone and protein concentrations and increased numbers of atretic follicles in the offspring of adult female rats ( 3 ) . nicotine has a direct effect on the ovaries and morphology of treated adult female rats ( 6 ) . administration of nicotine in adult female rats has resulted in the increase in the number of atretic follicles in the ovary , irregularities in the estrous cycle , impaired ovulation , altered steroid hormone concentration , decrease in the number and size of graafian follicles and corpora lutea ( 1 , 3 ) . nicotine exposure during fetal , neonatal ( 8) and adult hood of female rats ( 6 , 8) induces apoptosis in the ovaries . generally , there are few options to protect the ovarian function in females against side - effects of drugs , chemotherapy or radiotherapy . these include transposition of the ovaries outside the fields of radiation , administration of gonadotrophin releasing hormone ( gnrh ) analogs and cryopreservation of parts of the ovarian cortical tissue . most of these options are either ineffective or still belong to the field of research ( 9 ) . drugs that could protect the oocyte and its surrounding feeder cells from such damages would be very useful . it is the chief secretory product of the pineal gland and a powerful free radical scavenger and antioxidant ( 10 ) . melatonin facilitates various physiological functions as follows : circadian rhythm functions , such as sleeping and waking up ; sexual activity and reproductive functions ; tumor growth ; immune response ; and aging ( 11 ) . furthermore , due to its low toxicity , melatonin is used as a pharmacological substance for treating sleep disorders ( 12 ) . melatonin receptors have been detected in several organs , including brain , retina , cardiovascular system , gastrointestinal tract , kidney , immune cells , adipocytes , prostate and breast epithetlial cells , ovary granulosa cells , myometrium and skin ( 13 ) . a better understanding of the effects of nicotine is critical for women who are unable to quit smoking use nicotine replacement therapy ( nrt ) for cessation of smoking and its related side  effects . in comparison with smoking , nrt reduces exposure to thousands of toxic chemicals in the cigarette smoke ( 4 ) . the purpose of this study was to investigate the protective effects of melatonin on mouse ovary and follicogenesis following exposure to nicotine . a total of 32 adult nmri female mice were obtained from the razi institute in karaj , iran and they were randomly divided into four groups ( n = 8) . the animals were housed in small groups under standard lighting conditions with free access to water and food . they were allowed to adapt for at least one week in the animal room before they were subjected to treatment . animals were maintained and handled according to the protocols approved by the guilan university of medical sciences animal care and use committee . the assigned groups were as follows : group 1 ( controls ) : the mice received 1% ethanol ( 0.3 ml ) in normal saline intraperitoneally for 15 consecutive days.group 2 ( nicotine ) : the mice received 40 g / kg body weight nicotine ( sigma chemical company , st . louis , usa ) intraperitoneally for 15 days.group 3 ( melatonin ) : the mice received melatonin ( sigma , usa ) that had been dissolved in ethanol and further diluted in saline to give a final concentration of 1% ethanol ; a dose of 10 mg / kg was administered intraperitoneally for 5 consecutive days.group 4 ( nicotine and melatonin ) : the mice received 40 g / kg body weight nicotine ( sigma , usa ) and 10 mg / kg melatonin for 5 days . group 1 ( controls ) : the mice received 1% ethanol ( 0.3 ml ) in normal saline intraperitoneally for 15 consecutive days . group 2 ( nicotine ) : the mice received 40 g / kg body weight nicotine ( sigma chemical company , st . group 3 ( melatonin ) : the mice received melatonin ( sigma , usa ) that had been dissolved in ethanol and further diluted in saline to give a final concentration of 1% ethanol ; a dose of 10 mg / kg was administered intraperitoneally for 5 consecutive days . group 4 ( nicotine and melatonin ) : the mice received 40 g / kg body weight nicotine ( sigma , usa ) and 10 mg / kg melatonin for 5 days . we chose 40 g / kg nicotine for a duration of 15 days based on the previous studies on mouse and rat ( 6 , 14 ) ; in addition , we used different doses of nicotine ( 0.1 , 0.2 , 0.4 and 0.6 mg / kg ) on both ovary and testis in our pilot study ( 15 ) . a dose of 0.6 mg / kg nicotine killed most of the animals , while a dose of 0.1 mg / kg did not have any effects on ovaries . in humans it has been shown that shortly after smoking several cigarettes , the mean peak of nicotine in blood is about 50 ng / ml which is equivalent to 20 cigarette / day based on the dose of 40 g / kg nicotine ( 14 ) . another study showed that the role of melatonin on the cells is both time- and dose - dependent ( 16 ) . the dose and timing of melatonin administration were selected according to previous studies in which the antioxidant action of this agent was apparent ( 17 , 18 ) . all animals were dissected on the 16th day after initiation of the treatment . ovaries were removed , fixed in 10% neutral buffered formalin , dehydrated , and embedded in paraffin . later , 5 m sections were prepared followed by staining for histological and morphometrical assessments . blood samples were collected through the inferior vena cava , immediately after sacrificing the mice . the serum was separated and stored at -80  c. serum estradiol concentrations were measured using elisa kits ( demeditec diagnostics gmbh , germany ) . for this purpose , 5 m sections were stained by periodic acid schiff ( pas ) and observed using a standard light microscope . follicles were classified based on their morphological characteristics as : primordial , primary , secondary and graafian follicles . they were identified by the flattened granulosa cells surrounding the oocyte ( figure 1 ) . photomicrographs of mouse ovary ; a : primordial and primary follicles ; b : a primary and secondary follicle ; c : two secondary follicles ; d : a graafian follicle with a large antral cavity ; o : oocyte ; gl : granulosa layer ; tl : techa layer and a : antral cavity . bar : 50 micron they were identified by one layer of cuboidal granulosa cells surrounding the oocyte ( figure 1 ) . they were characterized by two or more layers of cuboidal granulosa cells or with presence of one or more cavity inside the granulosa cells with no visible antrum ( 1921 ) ( figure 1 ) . they were identified by the presence of a large antral cavity filled with secretory fluid ( 1921 ) , ( figure 1 ) . in each sample , all types of follicles were counted and measured using a graded ocular lense calibrated on an olympus light microscopy ( japan ) with 400 magnification . the data were analyzed by the analysis of variance ( anova ) and tukey post hoc tests . a total of 32 adult nmri female mice were obtained from the razi institute in karaj , iran and they were randomly divided into four groups ( n = 8) . the animals were housed in small groups under standard lighting conditions with free access to water and food . they were allowed to adapt for at least one week in the animal room before they were subjected to treatment . animals were maintained and handled according to the protocols approved by the guilan university of medical sciences animal care and use committee . the assigned groups were as follows : group 1 ( controls ) : the mice received 1% ethanol ( 0.3 ml ) in normal saline intraperitoneally for 15 consecutive days.group 2 ( nicotine ) : the mice received 40 g / kg body weight nicotine ( sigma chemical company , st . louis , usa ) intraperitoneally for 15 days.group 3 ( melatonin ) : the mice received melatonin ( sigma , usa ) that had been dissolved in ethanol and further diluted in saline to give a final concentration of 1% ethanol ; a dose of 10 mg / kg was administered intraperitoneally for 5 consecutive days.group 4 ( nicotine and melatonin ) : the mice received 40 g / kg body weight nicotine ( sigma , usa ) and 10 mg / kg melatonin for 5 days . group 1 ( controls ) : the mice received 1% ethanol ( 0.3 ml ) in normal saline intraperitoneally for 15 consecutive days . group 2 ( nicotine ) : the mice received 40 g / kg body weight nicotine ( sigma chemical company , st . group 3 ( melatonin ) : the mice received melatonin ( sigma , usa ) that had been dissolved in ethanol and further diluted in saline to give a final concentration of 1% ethanol ; a dose of 10 mg / kg was administered intraperitoneally for 5 consecutive days . group 4 ( nicotine and melatonin ) : the mice received 40 g / kg body weight nicotine ( sigma , usa ) and 10 mg / kg melatonin for 5 days . we chose 40 g / kg nicotine for a duration of 15 days based on the previous studies on mouse and rat ( 6 , 14 ) ; in addition , we used different doses of nicotine ( 0.1 , 0.2 , 0.4 and 0.6 mg / kg ) on both ovary and testis in our pilot study ( 15 ) . a dose of 0.6 mg / kg nicotine killed most of the animals , while a dose of 0.1 mg / kg did not have any effects on ovaries . in humans it has been shown that shortly after smoking several cigarettes , the mean peak of nicotine in blood is about 50 ng / ml which is equivalent to 20 cigarette / day based on the dose of 40 g / kg nicotine ( 14 ) . another study showed that the role of melatonin on the cells is both time- and dose - dependent ( 16 ) . the dose and timing of melatonin administration were selected according to previous studies in which the antioxidant action of this agent was apparent ( 17 , 18 ) . all animals were dissected on the 16th day after initiation of the treatment . ovaries were removed , fixed in 10% neutral buffered formalin , dehydrated , and embedded in paraffin . later , 5 m sections were prepared followed by staining for histological and morphometrical assessments . blood samples were collected through the inferior vena cava , immediately after sacrificing the mice . the serum was separated and stored at -80  c. serum estradiol concentrations were measured using elisa kits ( demeditec diagnostics gmbh , germany ) . for this purpose , 5 m sections were stained by periodic acid schiff ( pas ) and observed using a standard light microscope . follicles were classified based on their morphological characteristics as : primordial , primary , secondary and graafian follicles . they were identified by the flattened granulosa cells surrounding the oocyte ( figure 1 ) . photomicrographs of mouse ovary ; a : primordial and primary follicles ; b : a primary and secondary follicle ; c : two secondary follicles ; d : a graafian follicle with a large antral cavity ; o : oocyte ; gl : granulosa layer ; tl : techa layer and a : antral cavity . hematoxiline and pas staining . they were identified by one layer of cuboidal granulosa cells surrounding the oocyte ( figure 1 ) . they were characterized by two or more layers of cuboidal granulosa cells or with presence of one or more cavity inside the granulosa cells with no visible antrum ( 1921 ) ( figure 1 ) . they were identified by the presence of a large antral cavity filled with secretory fluid ( 1921 ) , ( figure 1 ) . in each sample , all types of follicles were counted and measured using a graded ocular lense calibrated on an olympus light microscopy ( japan ) with 400 magnification . the data were analyzed by the analysis of variance ( anova ) and tukey post hoc tests . ovaries were surrounded by a layer of simple squamous germinal epithelium . in the ovarian cortex , there were different types of follicles with various sizes . medulla was occupied by many blood and lymph vessels and a regular zona pellucida was observed around the oocytes in primary follicles onward . serum estradiol concentration in the control group was 60.2610.07 pg / ml ( figure 2 ) . photomicrographs of mouse ovary in different groups ; a : control mouse ; b : nicotine treated mouse ; c : melatonin - treated mouse and d : nicotine + melatonin treated mouse . notice regression of ovary and decrease in number and size of follicles in nicotine - treated ovary ( b ) . in d , melatonin has improved the folliculogenesis in the ovary in comparison with nicotine - treated mouse . bar : 50 micron upon microscopic examination , degeneration of follicles and desquamation of geranulosa cells inside the antral cavity and irregular zona pellucida around the oocytes were observed in the nicotine treated group ( figure 2 ) . in some follicles there were vacuoles inside the granulosa layers and shedding of picnotic granulosa cells inside the antral cavity was evident . visibly , administration of nicotine for 15 days caused a significant decrease in the number of secondary and graafian follicles ( p < 0.05 ) in comparison with the controls . the number of corpus lutea decreased significantly in nicotine treated group in comparison with the controls ( p < 0.05 ) . additionally , nicotine reduced serum estradiol concentration in comparison with the controls ( 44.2116.49 vs. 60.2610.07 pg / ml , p < 0.05 ) , ( table 1 ) . the effects of nicotine and melatonin on the number of mouse ovarian follicles and serum estradiol level the values are expressed as ( meansd ) . the values are comparable in the same row significant from control group ( p < 0.05 ) significant from nicotine - treated group ( p < 0.05 ) ; f ( follicle ) in contrast to nicotine , melatonin had no significant effects on estradiol concentrations ( 53.307.12 vs. 60.2610.07 pg / ml ) or ovarian follicles ( table 1 and figure 2 ) . co - administration of nicotine and melatonin significantly increased number of secondary and graafian follicles and corpus luteum in compareson with the nicotine only treated mice ( table 1 ) . serum estradiol concentrations significantly increased in group 4 ( 55.2212.43 vs. 44 .2116.49 pg / ml ) in comparison with group 2 ( table 1 and figure 2 ) . the present study showed that nicotine causes histopathological changes in the ovaries of mice , however , co - administration of melatonin and nicotine improved these alterations by providing protection against the impairment of follicogenesis partly through effect on hypothalamo - pituitary gonadal axis . results from in vivo and in vitro studies have clearly demonstrated that nicotine alone can have adverse effects on ovarian function ( 22 ) . similar to our study , it has been shown that nicotine exposure has a direct effect on the ovarian morphology of the treated female mice ( 6 ) . in the nicotine treated group , . this condition may be due to the increased effect of nicotine on granulosa cells . the number of granulosa cells in large follicles is much more than in small follicles . in this regard , nicotine acetylcholine receptors ( nachr-2 ) have been identified on granulosa cells ( 8) . it has been shown that nicotine can alter ratio of bcl2 : bax and activate caspase 3 in different tissues through its nachr-2 and -7 receptors ( 23 , 14 ) . therefore , through this probable mechanism and by nachr-2 , nicotine can have adverse effects mostly on growing or graafian follicles ( 14 ) . the above mentioned alterations in ovary might be due to induction of apoptosis by nicotine . an increase in apoptotic granulosa cells was found in rats following fetal and neonatal exposure to nicotine ( 8) . through its interaction with specific nachrs , nicotine has been shown to induce apoptosis in multiple tissues ( 2528 ) . expression of nachrs-2 and nachrs-7 have been identified in both fixed ovarian tissue and in isolated granulosa cells ( 8) . therefore , one mechanism by which nicotine may have initiated a change in ovarian morphology and function is by directly causing ovarian cell apoptosis via the nachrs . through this mechanism , nicotine would affect growing and developing follicles but would also likely negatively affect the pool of primary follicles , reducing subsequent ovulation events ( 8) . cigarette smoke likely causes follicle loss in mice ovaries without affecting ovulation ( 3 ) . in contrast blackburn et al . demonstrated that nicotine causes lh - independent inhibition of ovulation in vivo and in vitro in pmsg - primed imamture female rats ( 29 ) . decreased estrogen level in nicotine treated group can probably be explained by its toxic effects on granulosa cell function or aromatase activity . moreover , nicotine has been shown to inhibit the induction of progester - one synthesis in cumulus cells by fsh and the production of other androgens by theca interna cells ( 14 ) . in contrast to our study , nicotine did not affect estradiol production by human granulosa cells ( 1 ) . however , gocze et al . ( 30 ) and bodies et al . ( 31 ) observed a slight increase in estradiol production by human granulosa cells treated with nicotine , while barbieri et al . ( 32 ) reported decreased aromatase activity in these cells treated with aqueous tobacco smoke extract and nicotine alone . one reason for these conflicting results may be the variation in the form of used nicotine preparations . another reason could be the source of granulosa cells and the length of culture period and also species differences and dose of nicotine ( 1 ) . melatonin in dose of 10 mg / kg body weight daily for 5 days had no adverse effects on adult mouse ovary morphology . however , it caused significant increase in estradiol concentration in group 4 in comparison with the nicotine only treated group . melatonin exerts its primary reproductive action at the level of the brain and pituitary gland . however , the presence of high melatonin concentration in follicular fluid and the presence of receptors in granulosa cells suggest a potential beneficial property of melatonin on the ovarian function ( 5 ) . generally , melatonin toxicity is extremely low and no adverse effects have been found when 10 to 250 mg / kg melatonin has been fed to mice ; 100 to 250 mg / kg to rats or even 800 mg / kg to rabbits , dogs or cats . in human volunteers , no side effects were observed by the oral administration of melatonin in a dose of 1 to 300 mg or even 1 g daily for 30 days ( 33 ) . low doses of melatonin does not affect estrogen level in vitro , however , in higher doses melatonin reduces production of estrogen ( 9 ) . in other words , effect of melatonin on steroidogenesis is dose - dependent . probably , melatonin exerts its effect via a receptor - mediated action at the level of the ovary to modulate steroidogenesis ( 34 ) , and possibly luteolysis because it has been shown that melatonin mt1 and mt2 receptors are expressed in human granulosa lutein cells too . melatonin also regulates luteinizing hormone receptors ( lhr ) , gonadotropin releasing hormone ( gnrh ) and gonadotropin releasing hormone receptor ( gnrhr ) ( 34 ) . contrary to our expectation , melatonin was not effective on the number of follicles . in hamsters , melatonin causes decreased number of graafian follicles and corpora lutea and a proliferation of interstitial tissue in the ovary . hence , the exogenous melatonin may have an inhibitory , a stimulatory or no effect on the reproductive system of rodents depending on the model system and species used ( 35 ) and in humans , the effect of exogenous melatonin is different in the menstrual phase ( 36 ) . showed melatonin could increase lh receptors and secretion of progesterone in human luteal granulosa cells in cell culture ( 34 ) , however , bodies et al . showed reduction in the secretion of estradiol and progesterone in human luteal granulosa cells in cell culture ( 37 ) . in webley 's study , melatonin did not alter estradiol concentration ( 38 ) . it seems these conflicts depend on the cell type ( theca or granulosa cells ) , duration of treatment , experimental model ( cell or follicle cultures ) , species and dose of melatonin ( 39 ) . we showed that administration of melatonin in nicotine - treated mice protects follicogenesis in the ovary . it may also protect testicular tissue against busulfan , cisplatin and x - ray , cardiac muscle cells from damage induced by doxorubicin , and intestinal organ injury following mesenteric ischemia / reperfusion ( 40 ) . the other protective mechanisms of melatonin on ovary in nicotine - treated mice may be due to antioxidant activity of melatonin ; although , we did not measure the antioxidant property of melatonin . estrogen stimulates the proliferative activity of granulosa cells via fsh receptors . in the present study , however petric et al . did not find antiproliferative activity of nicotine on granulosa cells ( 8) . on the other hand , it has been shown in previous studies that melatonin has antiproliferative activity on male germ cells ( 40 ) . antiproliferative effects of melatonin using mcf7 cells as a model to study the anti - estrogenic effect of this hormone has been shown in several studies ( 41 , 42 ) . in conclusion , this study indicates that melatonin improves the ovarian functions in nicotine - treated mice through alterations in estrogen concentration . our results also suggest that melatonin may have a significant beneficial effect for clinical applications and subfertility or infertility induced by smoking in women .
backgroundnicotine exposure causes impaired fertility and ovarian dysfunction . 
 the aim of this study was to investigate the possible protective role of melatonin , which is known as an antioxidant agent on altered ovarian functions upon nicotine exposure.methodsa total of 32 female adult nmri mice were divided randomly into four groups ( n = 8) . 
 the control group received vehicle , while group 2 received nicotine ( 40 g / kg ) for 15 days and group 3 melatonin ( 10 mg / kg ) for 5 days . 
 group 4 received both nicotine ( 40 g / kg ) and melatonin ( 10 mg / kg ) for the same periods . 
 all animals were treated intraperitoneally . 
 after autopsy on the 16th day , histopathological and morphometrical examinations were performed and serum estradiol concentrations were measured . 
 the data were analyzed using anova and tukey post hoc test . 
 a value of p < 0.05 was considered significant.resultsnicotine significantly reduced the number of pre - antral and antral follicles , as well as estradiol concentration compared to the control group ( p < 0.05 ) . 
 however , the decrease in the number of primordial follicles was not significant in the nicotine treated group . 
 a significant increase in the atretic follicles were observed in group 2 compared to the control group ( p < 0.05 ) . 
 moreover , melatonin caused a marked normalization in the number of ovarian follicles and estradiol levels in group 4 compared to group 2.conclusionthe results from this study suggest that melatonin may have a protective effect against nicotine - induced ovarian changes on the number of different stages of follicle growth .