Study On Mechanisms Of Smoking On Male’s Reproductive Toxicity In Ratss

Objective: To research the effect of smoking on hypothalamic-pituitary-testicularaxis hormones and the toxic effects on testicular cells in rat, and analysis of the possiblemechanisms by related apoptosis gene. To study of cigarette smoke extract on testicularcell cytotoxicity and analysis of the possible mechanisms Methods:80SD rats wererandomly divided into8groups. Four groups were control group (C), and the others wereexposed groups, which was inhaled cigarette smoke for2h per day and continued for8weeks. The smoking group and the compared were respectively divided into4sub-groups,i.e. sub-group of2weeks inhalation, sub-group of4weeks inhalation, sub-group of6weeks inhalation, sub-group of8weeks inhalation and the reproduction group with10male mice in each group. By the end of2ed4th6th8thweek10male mice in the smokinggroup and the compared group were respectively killed and the index were observed. Thetestosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH) andinsulin-like growth factor-1(IGF-1) in rat serum and gonadotropin-releasing hormone(GnRH) in rat hypothalamus were determined by radioimmunoassay kit. The testicularorganization structure were observed with HE staining, The apoptosis of testicular cellsof different groups was located and quantitated in morphologic and cellular levels bymethod of Methyl green-pyronine staining and temimal dexynucleotidyl tranferase-mdiated dUTP biotin nick end labeling (TUNEL). The Bcl-2、Bax、Fas、FasL expressionsin testis of different groups were located and quantitated by method of Western blotting.Each group the level of Fas, Fas-L, Bcl-2mRNA expression were measured by RT-PCR.It was studied that the effect of cigarette smoke extract (CSE) on the proliferation andgrowth of pdmarily cultured Sertoli cells, Leyding cell and Spermatogenic cells in vitro.The three cells were exposed to different concentrations of CSE for12hours, which weredivided into three groups according to its concentration, including0%CSE group、 5%CSEgroup10%CSE group. Lactate dehydrogenase (LDH) was detected bybiochemistry, cell proliferation by MTT assay and CSE induced cell apoptosis wasobserved by Flow Cytometry. Results:1) With the extension of exposed time of smoking,the increase of weight in the smoke group were apparently lower than those of thecompared group especially in the group of4th,6thand8thweeks inhalation (P＜0.05).2)The testosterone in the smoking group significantly lower than the control group after the4thweeks (P＜0.05) and it was reduced with the extension of exposed time (P＜0.05).3)LH in the control rat serum was obviously raised with time elapsed especially in thegroup of6thand8thweeks, while it in the smoking group with time extended declined.The difference of LH between the smoking group and the control group was alsosignificant (P＜0.05).4) FSH was stable in the rat serum of the control group and thesmoking group, and it was distinctly lowered since the second week of the smokinggroup than the control group (P＜0.05);5) IGF-1levels decreased with time. IGF-1insmoking group of the8thweeks was significantly lower than the2edweeks, the4thweekand the6thweek (P＜0.05), while the2edweek, the6thweek and the8thweek of thesmoking group were significantly lower than those in the control group (P＜0.05)6)Gonadotropin-releasing hormone of rat hypothalamus either the control group or thesmoking group increased with time. GnRH in the smoking group was not statisticallysignificant than the control group (P＞0.05);7) The result of HE stain was that smokingcan lead to the testis tissue destruction, and organizational change was deepened withtime. The testis in the8thweek of the smoking group smoking obvious atrophy, a largenumber of cell lysis significant degenerative changes in the seminiferous tubules andinterstitial tissue;8) The result of TUNEL was that apoptosis index of the cells in thetestis of the4th,6thand8thsmoking group significantly higher than the control (P＞0.05);9) Fas/FasL and Bax protein in rat testis of the smoking group was raised while the Bcl-2protein was decresed. The mRNA expression of Fas/FasL, Bax was increased, the sameresults with protein expression. The gene expression of Fas/FasL, Bax in the smoking8thweeks t Fas/FasL, Bax was increased significantly (P＜0.05) and Bcl-2mRNA waslowered significantly (P＜0.05);10) the cell proliferation of the Leyding cell andSpermatogenic cells with the increased CSEs’ concentration was decreased significantly(P＜0.05) and appeared a dose-response relationship; LDH of the Sertoli cells, theLeyding cells and the Spermatogenic cells in CSEs group was significantly higher thanthat in the control group (P＜0.05);11) The result measured by flow cytometry showedthat CSE leaded to the apoptosis rate of the Sertoli cells, the Leyding cells and the Spermatogenic was significantly increased (P＜0.05). Conclusion: Smoking is harmfulfactors on the development of pubertal rats reproductive system, and have an adverseeffect on not only the Hypothalamic-pituitary-Testicular axis hormones but also testiculartissue and cells. The activation apoptosis regulatory genes by smoking leaded to acorresponding increase in protein expression, while inhibition of the protective proteinexpression. Chemicals from cigarette smoke extract resulted in Sertoli cells, Leyding celland Spermatogenic cells apoptosis. It suggested that smoking may lead to growthretardation on development of reproductive system in pubertal rat, and reduction in spermcount and function.