Effect of Cigarette Smoke Exposure on Kisspeptin Levels in Pubertal Female Rats: Role of Vitamin D Supplementation

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1 Med. J. Ciro Univ., Vol. 83, No. 2, September: 87-95, Effect of Cigrette Smoke Exposure on Kisspeptin Levels in Pubertl Femle Rts: Role of Vitmin D Supplementtion MAHA M. ATTIA, Ph.D. The Deprtment of Physiology, Medicl Reserch Institute, Alexndri University Abstrct Bckground: Animl nd humn studies hve shown ssocitions between ctive smoking nd ltered femle fertility. However, the reproductive effects of pssive smoking is reltively new compred to the effects of ctive smoking. Pubertl exposure to cigrette smoke my be ssocited with reproductive effects in dulthood. There re some evidence tht in ddition to sex steroid hormones, the clssic regultors of humn reproduction; Vitmin D (Vit D) lso modultes reproductive processes in women nd men. Recently, the neuropeptide kisspeptin hs been identified s potentil regultor of developmentl chnges in reproduction. Aim: The present study ws designed to evlute the effect of cigrette smoke exposure on kisspeptin levels, Hypothlmic-Pituitry-Gondl (HPG) xis in pubertl femle rts. The effect of Vit D supplementtion ws lso studied. Mteril nd Methods: Thirty femle pubertl lbino rts ged (35-40 dys) were divided into three groups: Group (I) 10 rts served s control, Group (II) 10 rts exposed to cigrette smoke (1 hour/dy) (CSE) nd Group (III) 10 rts supplemented with Vit D in dose of (650 IU/Rt/Dy) long with Cigrette Smoke Exposure (CSE +Vit D). In dult femle rts (85-90 dys), serum levels of Vit D, kisspeptin, Gondotropin Relesing Hormone (GnRH), Follicle Stimulting Hormone (FSH), Luteinizing Hormone(LH), estrogen nd progesterone were detected. Ovrin levels of estrogen, progesterone, totl ntioxidnt cpcity nd poptotic mrker (ctive cspse-3) were lso determined. Results: Cigrette smoke exposure cused significnt reduction in both serum levels of Vit D, Kisspeptin nd hypothlmic-pituitry-ovrin hormones (GnRH, FSH, LH, estrogen nd progesterone) nd ovrin levels of estrogen, progesterone nd totl ntioxidnt cpcity. Significnt elevtion in ctive cspse-3 ws observed. On the contrry, Vit D supplementtion cused significnt elevtion in serum levels of kisspeptin, hypothlmic-pituitry-ovrin hormones nd ovrin levels of estrogen, progesterone nd totl ntioxidnt cpcity. However, significnt reduction in poptotic mrker (cspse-3) ws detected. Kisspeptin ws positively correlted with Vit D in (CSE) nd (CSE +Vit D) groups nd with ovrin estrogen in (CSE) group p< On the other hnd its inversely correlted with ctive cspse-3 in both (CSE) nd (CSE + Vit D) groups. Correspondence to: Dr. Mh M. Atti, The Deprtment of Physiology, Medicl Reserch Institute, Alexndri University Conclusion: According to the findings of the present study, it cn be concluded tht exposure to cigrette smoke during pubertl period my led to reduction in Vit D. This could impir kisspeptin level, inducing hypothlmic dysfunction, nd ffect pituitry nd ovrin function. On the other hnd, vitmin D supplementtion modultes kisspeptin function which preserves hypothlmic-pituitry-gondl xis, prevents oxidtive dmge, loss of ovrin function nd reduces ovrin poptosis. Therefore, we could suggest tht Vit D my hve n importnt protective role ginst deleterious effects of cigrette smoke exposure. It could be considered s key regultor of neuroendocrine nd ovrin function. Key Words: Cigrette smoke exposure Kisspeptin Hypothlmic-pituitry-gondl xis Vitmin D supplementtion. Introduction CIGARETTE smoking is the most preventble cuse of morbidity nd premture mortlity worldwide. Smoking introduces wide rnge of diseses including, but not limited to, mny types of cncer, crdiovsculr diseses nd respirtory diseses [1]. Although, niml nd humn studies hve shown ssocitions between ctive smoking nd ltered femle fertility nd embryo development [2], the reproductive effects of secondhnd smoke exposure is reltively new compred to the effects of ctive smoking. Secondhnd Tobcco Smoke (STS), pssive smoking lso known s environmentl tobcco smoke is mixture of over 4000 chemicls, more thn 60 of which re known or suspected crcinogens or reproductive toxicnts (e.g. crbon monoxide, cdmium, led, benzene, nicotine, rdioctive polonium-210 nd Polycyclic Aromtic Hydrocrbons (PAHs) which were found to be higher in Sidestrem Smoke (SS) thn minstrem smoke (MS) (bout 10-fold) [3]. Some studies hve indicted significntly higher levels of smoking toxicnts in reproductive tissues or fluids thn in serum, which suggested tht the toxicnts ccumulted in the reproductive orgns [4]. 87

2 88 Effect of CSE on Kp Levels in Pubertl Femle Rts: Role of Vit D Supplementtion Non-smoking women who re exposed to second-hnd smoke re t incresed risk of difficulty becoming pregnnt, of giving birth premturely of stillbirth [5], of spontneous bortion [6], nd of hving bby with congenitl mlformtions [3]. Moreover, femle fertility cn be dmged in utero if the womn's mother ws exposed to secondhnd smoke while pregnnt [7]. There is lso new evidence tht developmentl exposures to tobcco smoke my be ssocited with reproductive effects in dulthood [8]. However, the complex processes by which cigrette smoke exposure my deleteriously ffect physiologicl mechnisms underlying sexul function is not clerly understood. At puberty, fertility is initited by the pulstile secretion of gondotropin relesing hormone (Gn- RH) from smll number of neurons in the hypothlmus. Although GnRH neurons re criticl component of the reproductive xis, Kisspeptin (Kp) peptides hve been identified recently s vitl upstrem regultors tht integrte centrl nd peripherl signls with GnRH relese, thereby plying pivotl role in the control of reproduction [9]. Kisspeptin, hypothlmic peptide, coded by the KiSS 1 gene, is novel neuromodultor sensitive to sex steroid feedbck nd metbolic cues. Kisspeptin is now recognized s crucil regultor of the onset of puberty. Kisspeptin expression hs been identified in multiple tissues, including pncres, dipose tissue, gonds, nd plcent, however, its min functionl role is medited by its expression within the centrl nervous system [10]. During the lst decdes, the outlook on vitmin D hs widened, from being vitmin solely involved in bone metbolism nd clcium homeostsis, to being multifunctionl hormone known to ffect brod rnge of physiologicl processes [11].There is some evidence tht in ddition to sex steroid hormones, the clssic regultors of humn reproduction, Vit D lso modultes reproductive processes in women nd men. It hs been reported tht Vitmin D Receptor (VDR) knockout mice hve significnt gondl insufficiency, decresed sperm count nd motility, nd histologicl bnormlities of testis, ovry nd uterus. Experimentl studies hve demonstrted tht ovry is trget orgn for Vit D rising the possibility tht vit D might ply role in modulting ovrin ctivity [12].However, the mechnisms by which vitmin D regultes femle reproduction is minimlly understood. Therefore, the present study ws designed to evlute the effect of cigrette smoke exposure on kisspeptin levels, Hypothlmic-Pituitry-Gondl (HPG) xis in pubertl femle rts. The effect of Vit D supplementtion ws lso studied. Mteril nd Methods Ethics sttement: This study ws pproved by the Ethics committee of the Medicl Reserch Institute, Alexndri University. Experimentl nimls: Thirty new born femle lbino rts were obtined from the niml house of Medicl Reserch Institute, Alexndri University, Egypt. Rts were mintined in polycrbonte cges t 22 ±2ºC on 12-hour light-drk cycle nd were provided with food, nd tp wter d libitum throughout the experiment. Visul inspection of the vulv ws performed until detection of vginl opening, the externl sign of puberty. Pubertl nimls (35-40 dys of ge) were divided into three groups: Group (I) 10 rts served s control, Group (II) 10 rts exposed to cigrette smoke for 1 hour/dy (pssive smoking) [13], Group (III) 10 rts exposed to the sme time of cigrette smoke long with Vit D3 supplementtion in dose of (650IU/rt/dy). The dult femle rts (85-90 dy of ge) were killed by decpittion, then trunk blood nd ovries were obtined from ll studied groups. Blood smples were collected nd centrifuged t 1500rpm for 15 minutes. Ser were liquoted nd Vit D levels were determined immeditely. Reminder ser were stored t 80ºC. Cigrette smoke exposure: Rts were plced in glss smoking chmber (closed box of 20cm length, 40cm bredth nd 25cm width). This box ws irtight, hving holes in two opposite sides nd ws covered by glss lid. A cigrette ws fixed into hole where the lit end ws introduced into the box nd the other end fixed in pump. Thus, by suction the cigrette smoke is introduced into the box. The type of cigrette used ws filter tipped contining 0.8mg of nicotine/cigrette. Preprtion of ovrin homogente: Ovries were dissected, clened from dhering mtter, their weights were recorded nd then homogenized in (0.015 M N2HPO4, 0.15 M NCl) buffer t PH 7.8 (10% homogente W/V) using Teflon utomtic homogenizer. The superntnt obtined from the homogente by ultrcentrifugtion nd filtrtion ws kept t 80ºC until biochemicl determintion.

3 Mh M. Atti 89 Biochemicl ssys: Serum level of 25-OH Vitmin D ws determined by using ELISA Kit. Serum level of Gondotropin Relesing Hormone (GnRH) ws determined using double ntibody sndwich type of Enzyme-linked Immunosorbent Assy (ELISA) using kits purchsed from Kmiy Biomedicl Compny. Folliculr Stimulting Hormone (FSH) nd Luteinizing Hormone (LH) were determined using Electro-Chemiluminescence Immunossy (ECLIA) employing two different FSH nd LH specific monoclonl ntibodies nd kits purchsed from Roche Dignostic. Serum kisspeptin ws determined by using ELISA kit. Estrogen nd progesterone levels were determined in both serum nd ovrin homogente by using (ECLIA) ssy. Colorimetric method ws used to determine ovrin totl ntioxidnt cpcity [14]. Active cspse-3 ws determined in ovrin homogente by using ELISA kit. Sttisticl nlysis: Dt were nlyzed using Sttisticl Pckge for Socil Sciences (SPSS). Normlly distributed quntittive dt were expressed in men ±SD nd were compred using F test (ANOVA) nd Post hoc Lest Significnt Difference test (LSD) for pirwise comprison, while bnormlly distributed dt were expressed in medin, (Min-Mx) nd were compred using Kruskl Wllis test nd Post Hoc test ws ssessed using Mnn-Whiteny test. Person correltion ws lso performed between serum kisspeptin nd both of Vit D nd ovrin prmeters. For ll sttisticl tests, level of 5% ws considered significnt. Results Tble (1) shows body weights, serum levels of Vit D, kisspeptin, GnTR, FSH, LH, estrogen nd progesterone. In ddition to ovrin levels of estrogen, progesterone, totl ntioxidnt cpcities nd ctive cspse-3 in the different studied groups. Significnt reduction in the men vlues of body weigh of rts exposed to cigrette smoke (CSE) thn control nd Vit D supplemented groups (CSE+ Vit D) ws detected (p<0.001). Exposure to cigrette smoke cuse significnt reduction in serum level of Vit D from 29.1 ±6.19 to 23.77±2.97 in control group, however, orl supplementtion with Vit D elevted this level to 35.95±4.68 (p<0.001) Fig. (1). Serum levels of kisspeptin in the different studied groups re represented in Fig. (2). Significnt reduction in kisspeptin level ws detected in (CSE) group. However, its level ws significntly elevted in (CSE+Vit D) group thn both control nd (CSE) groups. Serum levels of GnRH, FSH, LH, estrogen nd progesterone were significntly lower in (CSE) group s compred to control (p<0.001). Ovrin estrogen, progesterone nd totl ntioxidnt cpcity were reduced in (CSE) group s compred to control (p<0.001). However, significnt elevtion in ctive cspse-3 ws detected in the sme group Figs. (3-6). Orl supplementtion with Vit D3 to cigrette smoke exposed femle rts cuse n elevtion in serum levels of FSH, LH, estrogen nd progesterone (hypothlmic-pituitry-ovrin hormones) p< In ddition, significnt elevtion in ovrin estrogen, progesterone nd totl ntioxidnt cpcity were detected. However, cspse-3 ws significntly reduced in the sme group s compred to control (p<0.001). Therefore, Vit D my hve protective role ginst deleterious effects of cigrette smoke exposure. Ovrin weights: Although, there were slightly decrese in ovrin weights in (CSE) group s compred to control nd (CSE+Vit D) this reduction ws not sttisticlly significnt. Tble (1): Serum nd ovrin biochemicl prmeters in the different studied groups. Control (Group I) (n=10) (CSE) (Group II) (n=10) (CSE+Vit D) (Group III) (n=10) Serum Vit D (ng/ml) 29.10± ± b±4.68 <0.001* Serum kisspeptin (pg/ml) ( ) ( ) b ( ) <0.001* GRH (pg/ml) 19.49± ± b± 1.62 <0.001* FSH (ng/ml) 15.18± ± b±2.03 <0.001* LH (ng/ml) 5.62± ± b± 1.39 <0.001* Serum estrogen (ng/ml) 40.5 ( ) 27.0 ( ) 52.50b ( ) <0.001* Serum progesterone (ng/ml) 7.74± ± b± 1.5 <0.001* Ovrin estrogen (ng/g tissue) ( ) 50.0 ( ) 104.0b ( ) <0.001* Ovrin progesterone (ng/g tissue) 18.60± ± b±2.03 <0.001* T. ntioxidnt cpcity (mm/g tissue) ( ) 34.0 ( ) 61.5b ( ) <0.001* Ovrin cspse-3 (ng/g tissue) 1.65± ± b±0.25 <0.001* Normlly distributed quntittive dt ws expressed in men ± SD nd ws compred using F test (ANOVA) nd post Hoc test (LSD) for pirwise comprison, while bnormlly distributed dt ws expressed in medin (Min.-Mx.) nd ws compred using Kruskl Wllis test nd ws ssessed using Mnn-Whitney Test. : Significnt with Group I. b : Significnt with Group II. *: Sttisticlly significnt t p p

4 90 Effect of CSE on Kp Levels in Pubertl Femle Rts: Role of Vit D Supplementtion Men of Vit D (ng/ml) b Fig. (1): Vitmin D levels (ng/ml) in the different studied groups. Serum kisspeptin (pg/ml) b Fig. (2): Serum kisspeptin levels (pg/ml) in different studied groups. Ovrin estrogen (ng/g tissue) b Fig. (3): Ovrin estrogen levels (ng/g tissue) in different studied groups. Ovrin progesterone (ng/g tissue) Fig. (4): Ovrin progesterone levels (ng/g tissue) in different studied groups. b Ovrin totl ntioxidnt cpcities (mm/g tissue) b Ovrin ctive cspse-3 (ng/mg protein) b 20.0 Fig. (5): Ovrin totl ntioxidnt cpcities (mm/g tissue) in the different studied groups. 0.0 Fig. (6): Ovrin ctive cspse-3 (ng/mg protein) in different studied groups. Correltion studies: Tble (2) shows correltions between serum kisspeptin levels nd Vit D, ovrin estrogen, progesterone, totl ntioxidnt cpcities nd cspse-3 in the different studied groups. Significnt positive correltions between kisspeptin nd Vit D in both (CSE) nd (CSE+VitD) were detected (p=0.001, <0.001) respectively. Serum kisspeptin levels were positively correlted with ovrin estrogen in (CSE) group ( r=0.863, p=0.001) nd with totl ntioxidnt cpcities in both (CSE) nd (CSE+Vit D) (r=0.749, p=0.013, r=0.963, p<0.001) respectively. There were negtive correltions between kisspeptin nd ovrin cspse-3 in (CSE) nd (CSE+Vit D) (r=0.988, p<0.001, r=0,902, p<0.001) respectively.

5 Mh M. Atti 91 Tble (2): Correltions between serum kisspeptin levels, Vit D nd different ovrin prmeters. Serum kisspeptin (pg/ml) Control Group I (CSE) Group II (CSE+Vit D) Group III r s p r s p r s p Vit D (ng/ml) * * <0.001 Estrogen (ng/g tissue) * Progesterone (ng/g tissue) Totl ntioxidnt cpcities (mm/g tissue) * * <0.001 Cspse-3 (ng/mg protein) * < * <0.001 r s : Spermn coefficient. *: Sttisticlly significnt t p Discussion It hs been reported tht cigrette smoke hrms the reproductive system in mny spects. It impirs every stge of the reproductive process nd ech prt of the reproductive system such folliculogenesis, steroidogenesis, embryonic development nd trnsport. Cigrette smoke compounds interct with different reproductive trgets, depending on individul sensitivities, the presence of other toxic substnces nd ccording to time, dose, type nd durtion of exposure [15]. In the present study, exposure to cigrette smoke during pubertl period cuse reduction in serum levels of Vit D nd kisspeptine, ltertion in hypothlmic-pituitry-gondl xis, disturbnce in ovrin hormones nd elevtion in ovrin oxidtive stress nd poptosis. In ccordnce with the results of the present study, Vidl et l., [16] suggested tht cigrette smoke is reproductive toxicnt tht my cuse deleterious effects on ovrin function nd sex steroid hormone levels. Smoke compounds disrupt steroidogenesis, leding to impirment of estrogen (E2) synthesis nd progesterone synthesis deficiency. Moreover, in smoking model, lower levels of Folliculr Stimulting Hormone (FSH), Luteinizing Hormone (LH) nd Gondotropin Relesing Hormone (GnRH) were noticed [17]. It is well known tht cigrette smoking cn hrm fertility, but the existing reserch hs trgeted primrily on ovrin follicles, embryos or sex hormones [18]. Further studies investigting the mechnisms underlying cigrette smoking nd fertility concerned the effects of the inherent toxicnt molecules on follicles: For exmple, Benzo[]pyrene (BP), component of cigrette smoke; cused few of ovrin follicles [1], rupture in the grnulose cells, mitochondril lekge [19], reduced oolemm fluidity, impired fertiliztion in dulthood nd ging of oocyte nd dysfunction. Mtsunw et l., [20] dded tht ctivtion of Aryl Hydrocrbon Receptor (AhR) by BP stimultes vitmin D3 ctbolism. Polycyclic Aromtic Hydrocrbons (PAHs) reduced numbers of primordil nd primry follicles in rts nd mice. Moreover, exposure to toxicologicl levels of (PAHs), results in follicle loss by poptosis [21]. Cdmium (Cd), hevy metl compound in cigrette smoke, is n endocrine disruptor which is involved in the impirment of steroidogenesis, it hs long biologicl hlf-life nd ccumultes over time in ovries. In porcine grnulos cell lines exposed to low doses of Cd, incresed genomic expression of P450 Side-Chin Clevge (scc) enzyme, involved in the conversion of cholesterol to pregnenolone. At high doses, Cd ws shown to inhibit P450scc expression, resulting in decresed E2 synthesis. Moreover, high doses of Cd led to grnulos cell necrosis [2,22]. In ddition to nicotine dmge on grnulose cells, which hs been reported s promoting cell poptosis [15]. In ccordnce with the result of the present study, Durn et l., [23] reported tht there is n increse in ovrin poptosis with smoking. On the contrry, Tuttle et l., [24] mentioned tht exposure to cigrette smoke does not increse the rtes of poptosis in the ovry, nd by suggesting tht there is n incresed rte of follicle recruitment. Cigrette smoke my hve detrimentl effects on oocyte through inducing oxidtive stress nd injuring grnulos cells. This ws explined by Siddique et l., [25] who reported tht toxic follicle environment incresed oxidtive stress with decrese in SOD ctivities, inducing bnorml intercellulr cross-tlk, meiosis impirment nd ctiv-

6 92 Effect of CSE on Kp Levels in Pubertl Femle Rts: Role of Vit D Supplementtion tion of cell deth pthwys. Oxidtive stress reflects serious consequences, for instnce, enzymtic inctivtion, DNA frgmenttion, nd irreversible dmge of mitochondril DNA, membrne lipids, nd proteins resulting in mitochondril dysfunction nd ultimtely cell deth. In chroniclly Vit D deficient femles, incresed expression of genes responsible for enzymes involved in cellulr ntioxidnt defense systems suggest higher levels of intr-ovrin oxidtive stress. A decrese in Gsr expression, which is sulfur-redox cycle enzyme importnt in mintining blnced redox stte nd reduced free rdicl formtion, could lso contribute to the poor fertility outcomes seen in Vit D deficient popultions [26]. Other studies mentioned tht the effects of cigrette smoke on grnulos cells nd romtse enzyme decreses estrogen production, nd the lkloids in cigrette inhibit the production of progesterone [27]. It hs been estblished tht Vit D cts s regultor of number of enzymes involved in the regultion of steroid hormones production, nd thereby the production of both drenl steroid hormones nd sex hormones [28]. Vit D lters the romtse ctivity in plcentl cells [29] nd prostte cells [30]. Vitmin D receptor null mutnt mice hve decresed romtse ctivity in the ovry, testis nd epididymis. In brest cncer cell lines, Vit D tretment resulted in decresed romtse gene expression, while the sme tretment incresed the romtse gene expression in osteosrcom cell lines. Therefore, vitmin D3 hs been proposed to be tissue-selective romtse modultor [11]. Vitmin D deficiency my excerbte symptoms of Polycystic Ovrin Syndrome (PCOS), ssocited with ovultory nd menstrul irregulrities nd lower pregnncy success. This is not corrected by normlizing the hypoclcemi in Vit D-deficient femle rts, but requires vit D [32]. In the present study Vit D supplementtion long with cigrette smoke exposure reduced ovrin poptosis, protects ovrin tissues from oxidtive stress, increses kisspeptin nd then, regultes the reproductive xis. Kisspeptin ws positively correlted with vit D nd negtively correlted with poptotic mrker (ctive cspse-3) in both Cigrette Smoke Exposed (CSE) nd Vit D supplemented exposed (CSE+Vit D) groups. It hs been estblished tht, in norml tissues vitmin D plys n importnt role in promoting poptosis. It regultes poptosis ccording to the requirements of the body t different physiologicl stges [12]. Moreover, the expression of VDR mrna in the ovries, in mixed ovrin cells, nd in purified grnulos cell cultures indicting role of Vit D in steroidogenesis. In humn ovrin tissue, Vit D stimulted progesterone production by 13%, estrdiol production by 9% nd estrone production by 21% [11]. It hs been estblished tht sex steroids ply mjor role in regultion of serum kisspeptin level which in turn ffects fertility. The mjority of kisspeptin neurons express estrogen, progesterone nd ndrogen receptors, consistent with their role s meditors of sex steroid feedbck on reproductive xis [32]. In the present study positive correltion between ovrin estrogen nd serum kisspeptin ws detected in cigrette smoke exposed group only; however, no correltion ws detected in Vit D supplemented exposed group. This my be due to the direct effect of Vit D on kisspeptin level. Therefore, we cn suggest tht Vit D hs direct nd indirect (by enhncing estrogen level) effects on kisspeptin level. A recent study demonstrted tht E2 is essentil for the prepubertl development of kisspeptin peptide nd suggested tht n E2-kisspeptin positive feedbck mechnism exists before puberty. This implies kisspeptin neurons re E2-dependent mplifier of GnRH neuron ctivity in the prepubertl period. Estrdiol is responsible for inititing kisspeptin expression in periventriculr hypothlmic neurons tht re thought to ctivte gondotropin-relesing hormone neurons controlling puberty onset [33]. In support of this hypothesis, some investigtors demonstrted tht kisspeptin 1 neurons re virtully bsent in estrdiol-deficient, romtse knockout mice. Thus, it is possible tht the pubertl increse in Kiss1/Kiss1r mrna in femles is due to the increse in estrogens t this stge of development [34].In ddition, due to the regultory role of kisspeptin on GnRH neurons, it is thought tht kisspeptin my ply mjor role in pulse genertion. In rts, dministrtion of kisspeptin ntgonist directly into the rcute nucleus cused profound reduction in the number of LH pulses generted [35].Kisspeptin is principl regultor of the secretion of gondotropins, nd through this key role it is criticl for the onset of puberty, the regultion of sex steroid-medited feedbck nd the control of dult fertility [36]. Kisspeptin cells in the Arcute nucleus (Arc) pper to receive nd forwrd signls pplicble to negtive feedbck regultion of

7 Mh M. Atti 93 GnRH. In the femle rodent nteroventrl periventriculr nucleus (AVPV) kisspeptin cells re importnt for positive nd negtive feedbck regultion of GnRH nd their number in femle mice progressively increses until the ge of puberty [37,38]. Additionlly, it ppers tht in femle rodents n estrogen-dependent developmentl increse in kisspeptin peptide nd mrna in the AVPV occurs, leding presumbly to n increse in the secretory ctivity of this rostrl popultion of kisspeptin neurons. Physiologicl nd phrmcologicl studies indicting tht kisspeptin is the most potent GnRH secretgogue [32]. It ws mentioned tht Vit D regultes humn chorionic gondotropin expression nd secretion in humn syncytiotrophoblsts nd increses plcentl sex steroid production [39]. In humns, GnRH nd its receptor hve been found in extr-pituitry tissues such s the ovry, brest nd plcent. In the ovry, the expression of GnRH receptors hs lso been demonstrted in humn ovrin grnulos cells [40]. Moreover, kisspeptin hs role in reproductive neuroendocrine signling outside of the brin which suggested by the finding of both Kiss 1 nd Kiss1r in pituitry gondotrophs. Thus kisspeptin my regulte reproductive function t both hypothlmus nd pituitry level [41]. Other study demonstrted tht GnRH neurons express Vitmin D Receptor (VDR) protein, rising the possibility tht Vit D3 directly regultes GnRH neurons. Moreover, VDR re found in the gonds, hypothlmus, nd pituitry, suggesting tht the reproductive xis my be regulted by prcrine nd/or utocrine ctivities of Vit D [42]. Therefore, we propose tht Vit D deficiency most likely impirs femle reproductive function by inducing hypothlmic dysfunction, which secondrily ffects pituitry nd ovrin function. It ws reported tht, in the nervous system, the ctive Vit D ffects the conduction of the motor neurons nd synthesis of neurotrophic fctors, thus preventing dmges to the neurons. In ddition, Vit D reduces mrkers of oxidtive stress in vsculr nd nervous tissues. Additionlly, developmentl Vit D deficiency is hypothesized to dversely ffect neurodevelopment [43,44]. These dt suggest tht vitmin D is key regultor of neuroendocrine nd ovrin function. Finlly, findings of the present study indicte tht cigrette smoke is high risk for femle reproductive system. Exposure to cigrette smoke my cuse reduction in Vit D. This could impir kisspeptin level, inducing hypothlmic dysfunction, nd ffect pituitry nd ovrin function. On the other hnd, Vit D supplementtion modultes kisspeptin function which preserves the hypothlmic-pituitry-gondl xis. In ddition, Vit D reduces ovrin poptosis, prevents oxidtive dmge nd loss of ovrin function. This occurs through its direct nd indirect roles on kisspeptin. Therefore, we could suggest tht Vit D my hve n importnt protective role ginst deleterious effects of cigrette smoke exposure. We certinly recommend tht quitting exposure to cigrette smoke is wise choice to ensure good fertility. The determintion of optiml Vit D levels in the pubertl period nd the mount of vitmin D supplementtion required to chieve those levels for the numerous ctions of vitmin D throughout femle's life would hve importnt public helth implictions. References 1- CHRISTOPHER B., HARTE C.B. nd MESTON C.M.: The Inhibitory effects of physiologicl sexul rousl in nonsmoking women: Results from rndomized, Double- Blind, plcebo-controlled, cross-over tril. J. Sex. Med., 5: , DECHANET C., ANAHORY T., MATHIAU DAUDE J.C., et l.: Effects of cigrette Smoking on reproduction, Humn Reproduction Updte, 17 (1): 76-95, MOIR D., RICKERT W.S., LEVASSEUR G., et l.: A comprison of minstrem nd sidestrem mrijun nd tobcco cigrette smoke produced under two mchine smoking conditions. Chem. Res. Toxicol., 21: 4502, MEEKER J.D. nd BENEDICT M.D.: Infertility, pregnncy loss nd dverse birth outcomes in reltion to mternl secondhnd tobcco smoke exposure. Curr. Women's Helth Rev., 9 (1): 41-9, NIEUWENHUIJSEN M.J., DADVAND P., GRELLIER J., et l.: Environmentl risk fctors of pregnncy outcomes: A summry of recent metnlyses of epidemiologicl studies. Environ. Helth, 15 (12): 6, PEPPONE L.J. PIAZZA K.M., MAHONEY M.C., et l.: Associtions between dult nd childhood secondhnd smoke exposures nd fecundity nd fetl loss mong women who visited cncer hospitl. Tob. Control, 18 (2): , YE X., SKJAERVEN R., BASSO O., et l.: In utero exposure to tobcco smoke nd subsequent reduced fertility in femles. Hum. Reprod., 25 (11): AMIRKHANI J., YADOLLAH-DAMAVANDI S., MO- HAMMAD S., et l.: Correltion between Abortion nd Infertility mong Nonsmoking Women with History of Pssive Smoking in Childhood nd Adolescence. Interntionl Journl of Reproductive Medicine, , SKOUPORUPSKAIT E.K., GEORGE J.T. nd ANDER- SON R.A.: The kisspeptin-gnrh pthwy in humn

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