| In recent years,the incidence of male infertility showed increasing tendency worldwide.The main causes of male infertility include azoospermia,oligospermia,asthenospermia and teratozoospermia.However,the molecular mechanisms underlying male infertility is very complicated and yet to be elucidated.Sperm originate from the spermatogonia cells and then undergo a highly complex process involving mitosis,differentiation,migration,meiosis and spermiogenesis.Sperm produced in the testis are further transported across the efferent duct to the epididymis,in which possess fertilizing capacity by sperm maturation.These processes are regulated critically by numerous genes and proteins as well as noncoding RNAs,such as micro RNAs.Exploring and comprehending the molecular mechanism involved in spermatogenesis and sperm maturation will help to provide new idea and means for treating male infertility.In the first part of the thesis,the effect of two micro RNA clusters,mi R-34b/c and mi R-449,on male infertility were investigated.Firstly,we detected the expression of the mi RNAs in different tissues and found their high expression in tissues with motile cilia,such as efferent duct.Moreover,histological analysis of the mi R-34b/c-/-;mi R-449-/-(DKO)mouse displayed the loss of cilia in efferent duct,retardation of sperm in efferent duct and rete testis,severely atrophic and disorganized seminiferous epithelia.In order to explore the effect of mi R-34b/c and mi R-449 on spermatogenesis,efferent duct ligation was applied to simulate the obstruction of efferent duct,and the morphology of testis and efferent duct after ligation was similar to that in DKO mouse.Meanwhile,according to the results of efferent duct cell culture,mi RNA inhibition and immunofluorescent staining of cilia structure,we found that deficiency of mi R-34b/c and mi R-449 in efferent duct cells would cause the loss of cilia.Thus,all the results above indicated that the loss of cilia in efferent duct caused the obstruction of efferent duct and further increased the stress in seminiferous tubule,which finally impaired spermatogenesis and caused male infertility.On the other hand,we investigated the molecular mechanism of the compensation between mi R-34b/c and mi R-449.Using bioinformatics analysis,a transcription factor,STAT6,was found to be the target gene downregulated by mi R-34b/c and mi R-449,and at the same time,STAT6 potentially regulates the expression of the two mi RNA clusters.Then,the crosstalk between STAT6 and the two mi RNA clusters was confirmed by Ch IP-PCR,luciferase assay,STAT6 RNAi and overexpression tests in vitro.Moreover,it was showed that compensation between mi R-34b/c and mi R-449 was eliminated after repression of STAT6 in the cell.Thus,the results above indicated that STAT6 involved in the compensation between mi R-34b/c and mi R-449.In the second part of the thesis,we found for the first time that a transcription factor,TFEB,expressed in the spermatogonia of the testis.Then,the potential function of TFEB in spermatogonia was investigated.Using immunohistochemical staining,spermatogonia cell isolation and culture,we found the intensive expression of TFEB in the differentiating spermatogonia,which was regulated by retinoic acid(RA).Furthermore,the target genes regulated by TFEB in spermatogonia were investigated by Ch IP-seq test.The result showed that Axin2,Chl1,Dock2 and Nbea were regulated by TFEB,through which would affect the migration of spermatogonia,especially the migration across the junction of blood-testis barrier.In conclusion,this study demonstrated that mi R-34b/c,mi R-449 and TFEB play critically important functions during spermatogenesis,and provided novel insights into the underlying mechanism,which greatly contribute to illuminating the mechanism of male infertility and improving the clinical diagnosis as well as treatment of male infertility. |
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