Nicotine Induced Murine Spermatozoa Apoptosis Via Up-regulation Of Deubiquitinated RIP1 By Trim27 Promoter Hypomethylation

Smoking is one of the biggest public health problems worldwide.There are more than 6,000,000 people who die of smoking related diseases annualy,out of which more than 5,000,000 people smoke initiatively while more than 600,000 people take second-hand smoke.The smoking population in our country is more than 3 billion.There are 28.1% people over 15 that smoke,in which male take part of 52.9%.Apart from damage to the digestive and cardiovascular system through the sympathetic nervous system,nicotine is reported to directly impair male fertility.Research objectPrevious studies in the lab had figured out that smoking would result in reductions in the conventional parameters of semen,such as semen volume and sperm density.To further make clear the path through which smoking cause these damages,we researched the impact of nicotine on germ cell apoptosis and further researched the underlying molecular mechanisims.MethodsIn this study,we set up a nicotine-treated mice mode at first.We then used DNA ladder to detect the overall apoptosis level of the testis,and used TUNEL to further figure out the species of the apoptotic cells and there number.Transmission electron microscopy was then used to observe the ultra structures of elongating spermatids.Meanwhile,the active form of Caspase3 in sperm was also detected by Western blot.Bioinformation analysis was then conducted to figure out the functioning apototic pathway,which was then verificated by Western blot and RT-PCR.Bisulfate-sequencing was then used to carry out the promoter methylation analysis of differentially expressed genes.At last,we would conduct over-expression and si RNA inhibition of methylation regulated genes in spg GC-1 cells to verificate its function on the apoptotic pathway and thereafter cell apoptosis.ResultsNicotine significantly promoted apoptosis in stages Ⅰ,Ⅶ,Ⅷ,and Ⅺ spermatogonia,stages Ⅰ,Ⅶ,Ⅷ,Ⅹ,and Ⅺ spermatocytes,and stages I–Ⅴ,Ⅶ,and Ⅷ elongating spermatids.To explore the underlying molecular mechanisms,sperm m RNA next-generation sequencing of nicotine-treated mice was conducted.Out of the 86 genes related to apoptosis,Tnf(tumor necrosis factor alpha)was screened to be the most significant varied transcript,and the Onto-pathway analysis indicated that the TNF apoptotic pathway was especially activated by nicotine exposure.The TNF pathway was further studied at the gene and protein levels.The results showed that RIP1,the key component in the TNF apoptotic pathway,was up-expressed in its deubiquitinated form in nicotine-treated mice testis.TRIM27,an E3 ubiquitin ligase that activated TNF apoptotic pathway through up-regulating deubiquitinated RIP1,was also overexpressed in nicotine-treated spermatocytes;moreover,four consecutive Cp G sites near the Trim27 transcription start site were less frequently methylated.Finally,in vitro experiments of TRIM27 overexpression and RNA interference in GC-1 spermatogonial cells confirmed that the RIP1 deubiquitination and Trim27 hyopmethylation were both positively correlated with spermatocyte apoptosis.ConclusionIn summary,our study suggests that nicotine may induce murine spermatozoal apoptosis via the TNF apoptotic pathway through up-regulation of deubiquitinated RIP1 by Trim27 promoter hypomethylation.