Effects Of Cigarette Exposure On Reproductive System Of Male Mice And Protective Effect Of Molecular Hydrogen

BackgroundAccording to statistics,36% of men in the world are smokers.The total number of smokers in China has reached 320 million,and it is still growing at a rate of 2% per year.The number of people dying from tobacco-related diseases in China is nearly 1 million every year.At the same time,infertility affects nearly 10 to 15 percent of couples worldwide,with male factors accounting for 40 to 50 percent of infertility cases.Multiple studies have shown that harmful ingredients in cigarettes can damage the blood testosterone barrier,cause changes in testicular tissue structure,affect the production of sperm,and ultimately lead to damage to the male reproductive system,resulting in a decline in male fertility.In recent years,hydrogen,as a new、safe and effective antioxidant,has a wide application prospect with few side effects,and has become a research hotspot in various fields such as anti-oxidation and anti-apoptosis.Many animal studies and clinical trials show that hydrogen as a kind of small molecular substances,it can spread and penetrate the cell membrane,play a wide range of biological effects,the acute pancreatitis and sepsis,respiratory system disease,ischemia-reperfusion injury,disease,autoimmune diseases,organ transplantation and other acute or chronic inflammatory disease treatment and prevention effect.We were interested in whether molecular hydrogen could play a protective role in male reproductive system damage caused by cigarette exposure.The molecular mechanism of the damage to the male reproductive system caused by cigarette exposure is not fully understood.Single-cell transcriptome sequencing technology can reverse transcription,amplify and sequence all RNA of a single cell,and then conduct bioinformatics analysis.In this study,single-cell transcriptome sequencing was used to investigate the effects of cigarette exposure on mouse testicular germ cells and the possible damage mechanism.After preliminary experimental research,we have carried out the following discussion.Firstly,the effects of cigarette exposure on male reproductive system were determined,and an animal model of reproductive system injury caused by cigarette exposure was established.Secondly,molecular hydrogen was used for injury intervention to clarify the protective effect of molecular hydrogen on male reproductive system injury caused by cigarette exposure.Finally,single-cell transcriptome sequencing was used to investigate the effects of cigarette exposure on mouse testicular germ cells and the underlying biological mechanisms.Contents1.Effects of cigarette exposure on general condition,body weight and testicular weight of male mice2.Effects of cigarette exposure on reproductive system of male mice3.Protective effect of molecular hydrogen on reproductive system injury induced by cigarette exposure in male mice4.Single-cell transcriptome sequencing of mouse testicular tissueMethods1.Effects of cigarette exposure on general condition,body weight and testicular weight of male mice.During the whole experiment,the general growth status of each group of mice was observed,such as hair,drinking water,diet,mental state,excretion and activity.The weight change was recorded every 3 days,and the weight was weighed again before the mice were killed,which was beneficial to master the body weight change of the mice during the whole experiment.2.Effects of cigarette exposure on reproductive system of male miceHE pathological staining,Tunel staining,immunohistochemistry and other experimental methods were used to analyze the specific damage effect of cigarette exposure on testicular tissue of male mice.CASA was used to detect the sperm parameters of caudal epididymis of male mice,and the effects of cigarette exposure on sperm quality parameters were analyzed.The effects of cigarette exposure on sperm malformation rate in mice were analyzed by sperm modified Babbitt staining.3.Protective effect of molecular hydrogen on cigarette exposure induced reproductive system injury in male miceThe protective effects of molecular hydrogen on testicular tissue damage in male mice induced by cigarette exposure were analyzed by means of HE pathological staining,Tunel staining and immunohistochemical detection of PCNA.CASA was used to detect sperm parameters of caudal epididymides of male mice,and the protective effect of molecular hydrogen on the reduction of sperm quality parameters caused by cigarette exposure was analyzed,and the protective effect of molecular hydrogen on the abnormal sperm morphology caused by cigarette exposure was analyzed by sperm modified Babbitt staining.4.Single cell transcriptome sequencing was used to investigate the effects of cigarette exposure on mouse testicular germ cells and the underlying biological mechanisms.Results1.Effects of cigarette exposure on general condition,body weight and testicular weight in male mice.The body weight of mice in the control group increased steadily,their mental state was GOod,their hair color was healthy and shiny,their drinking water and diet were normal,their movements were quick and their excretion was normal.After cigarette exposure,the weight of mice decreased,their spirits were listless,their hair color was yellow,their drinking water and diet were reduced,their actions were slow,and their testicular weight was reduced after death.2.Effects of cigarette exposure on reproductive system of male mice.(1)Cigarette exposure resulted in the destruction of normal pathological structure of testicular tissue,atrophy and degeneration of testicular spermatogenic tubules,reduction of spermatogenic cell layers,and calcification foci.(2)Cigarette exposure increased the apoptosis rate of testicular tissue in mice(3)Cigarette exposure reduced the proliferation activity of testicular tissue cells to a certain extent,but there was no statistical significance.(4)Cigarette exposure decreased sperm concentration,motility and total sperm motility in caudal epididymis of mice,but had no significant effect on sperm malformation rate.3.Protective effect of molecular hydrogen on reproductive system injury induced by cigarette exposure in male mice(1)Molecular hydrogen effectively alleviated the decrease of body weight and testicular weight in mice.(2)Molecular hydrogen can effectively reduce the pathological structure damage of testicular tissue in mice after cigarette exposure.(3)Molecular hydrogen can effectively reduce the apoptosis of mouse testicular tissue after cigarette exposure.(4)Molecular hydrogen had no significant effect on the proliferation activity of testicular tissue cells induced by cigarette exposure.(5)Molecular hydrogen effectively alleviated the decline of sperm quality caused by cigarette exposure,improved sperm concentration,forward motility and total motility of male sperm,but had no significant effect on and malformation rate.4.Single cell transcriptome sequencing was used to investigate the effects of cigarette exposure on mouse testicular germ cells and the underlying biological mechanisms.(1)After single-cell suspension preparation and library construction of fresh testicular tissue,dimensionality was reduced by t SNE,and single-cell population was further divided by unsupervised clustering according to gene expression.Identification results based on the cell type of the population(stage of development)can be divided into 10 germ cell types,They were SSC,Diff-ing-SPG,Diff-ed-SPG,Pre-Leptotene,Leptotene-Zygotene,Pachytene,Diplotene,Rs,Es,Sperm.(2)To reconstruct the dynamic change process of cells over time,the intercellular dynamic change trajectory was constructed by using quasi-sequential analysis technology.The 1000 genes with the most significant difference between the control group and the cigarette exposure group were selected by Beam analysis,and the clustering of these genes was visualized to observe the changes of each expressed gene module in the quasitemporal process.These genes were enriched into GO function analysis and Pathway signaling Pathway analysis,respectively.Through analysis,it was found that in the early stage of spermatogenesis,functional analysis and signal enrichment analysis showed that the cellular biological processes affected by cigarette exposure mainly included cell cycle,cellular response stimulated by DNA damage,chromatin structure,cell division,ribosomal biosynthesis DNA repair,etc.In the middle stage of spermatogenesis,functional analysis and signal enrichment analysis showed that the cellular biological processes affected by cigarette exposure were related to mitotic spindle assembly,mitotic sister chromatid cohesion of centromeres,cell mitosis,sperm flagellum assembly,and cell cytokinesis regulation.At the late stage of spermatogenesis,functional analysis and signal enrichment analysis showed that the cellular biology affected by cigarette exposure was mainly related to the structure of sperm flagella,cilia and chromatin.(3)Through GO functional annotation and Pathway signaling Pathway analysis of cells at the early stage of spermatogenesis,it was found that cigarettes were highly enriched in spermatogenic tubule development,spermatocyte development,microtubule movement,cell proliferation and apoptosis during the early stage of spermatogenesis.Wnt signaling pathway,MAPK signaling pathway,cell cycle,pyruvate metabolism and other signaling pathways were significantly affected.(4)Through Qusage analysis,it was found that cigarette exposure resulted in a decrease in d-arginine metabolism compared with the control group,which may be the internal mechanism of cigarette influencing spermatogenesis.(5)The interaction between cells in mouse testicular tissue was revealed by cell communication technology,providing a new idea for the specific mechanism of testicular injury caused by cigarette exposure.ConclusionsCigarette exposure can reduce the body weight and testicular weight of male mice,cause pathological structure damage of testicular tissue,increase testicular cell apoptosis,decrease cell proliferation activity,and decrease sperm quality parameters.Molecular hydrogen can protect the germ cell damage caused by cigarette exposure,reduce the damage of pathological structure caused by cigarette exposure,reduce the apoptosis of testicular cells,restore cell proliferation to a certain extent,and also protect sperm quality parameters to a certain extent.For the control group and cigarettes exposed mice testis single-cell transcriptome sequencing found that cigarette can affect sperm production by promoting apoptosis,cell meiosis,inhibition of seminiferous tubule development,reducing DNA damage and arginine metabolism,influencing the Wnt signaling pathway、MAPK signal pathway,causing damage to the reproductive system,affecting the whole spermatogenesis process.