Effect Of Automobile Exhaust Exposure On Male Mice Spermatogenesis Function Mediated By NO-NOS Pathway

Objective :Through the production of automobile exhaust exposure device and establishment of animal model, we observe the effect of automobile exhaust exposure on spermatogenesis function of male mice, and explore the possible molecular mechanism about spermatogenesis function, and provide experimental evidence for the research on the reproductive toxicity of male mice. Increase people’s attention to environmental pollution caused by automobile exhaust, and enhance the awareness of environmental protection.Methods:1.Design and establishment of automobile exhaust exposure device, the automobile exhaust concentration in the device is verified to determine the stability of the automobile exhaust exposure condition.2. After exposure to automobile exhaust for 40 days, Spermatogenesis Function of male mice were observed: the quality of testis, epididymis and body mass were detected, and calculate the coefficient of viscera, epididymis sperm density, sperm motility, sperm motion parameters were detected by computer-assisted sperm analysis(CASA) detection system; preparation of testicular tissue pathological section, HE staining, the histological changes in the testes were observed by light microscope.3. The effect of the oxidation level in plasma and testis tissue of male mice were observed after automobile exhaust exposure. after blood was taken from the right ventricle, plasma was prepared by centrifugation, after testis homogenate, the protein was detected.the level of malondialdehyde(MDA) was detected by TBA method, and the level of total antioxidant capacity(T-AOC) was detected by ABST assay, and the level of NO was detected by Griess assay.4. The expression of e NOS,iNOS, P38 and P-P38 in the testis of male mice after automobile exhaust exposure were observed. The expression levels of eNOS,i NOS, P38 and P-P38 were detected by Western Blot method.Results:1.Constructed successfully the vehicle exhaust exposure device, through the ventilation test, The lethal time was 60 seconds, Half lethal time was 30 seconds. After 30 seconds the automobile exhaust is mixed, Gas ratio in the device( automobile exhaust: fresh air =1:10). After each ventilation, the mice were exposed to 1 hour, which was suitable for the exposure time and the model was stable. Through the detection of the internal components of the continuous exposure device, there was no obvious difference between the begin and the end of exposured in the device, the content of oxygen, the content of sulfur dioxide, the content of PM2.5 is no significant difference(p>0.05). Blood gas analysis results showed that there was no significant difference in PCO2, PH and PO2 between exposed and control groups(p>0.05), and no significant hypoxia was performed.2. There was no significant difference in the weight of testis, epididymis weight and organ coefficient between the two groups after exposure to automobile exhaust(P>0.05). The density and activity of sperm in the epididymis were decreased(P<0.05). The pathological changes of testis tissue in exposed group suggested that the arrangement of the germ cells were sparse, the sperm cells were decreased obviously, and the appearance of vacuole like changes, the sperm in the lumen was reduced.3. NO and MDA levels in plasma and testis of male mice after exposured by automobile exhaust were significantly increased, compared with the control group, with statistical significance(P<0.05), T-AOC levels in plasma and testis tissue were significantly decreased compared with the control group, with statistical significance(P<0.05). Compared with the control group, the expression of P38 protein in the exposed group had no obvious change, but the expression of P-P38, i NOS and eNOS are increased, Compared with the control group, ratio of eNOS/GAPDH、iNOS/GAPDH、p-p38/ GAPDH is increased in the exposed group, with statistical significance(P<0.01).but ratio of p38/ GAPDH is no statistical significance(P>0.05).Conclusion:1.Successful construction of automobile exhaust exposure experimental device and animal model.2.Under automobile exhaust exposure, spermatogenesis function of male mice is damaged.3.Oxidative stress in male mice is induced by vehicle exhaust, oxidative stress activated NO-NOS signaling pathway,the activation of this pathway may be one of the mechanisms of spermatogenesis dysfunction on male mice.