The Function Study Of Tbc1d8 Gene And MiR-92b In Gametogenesis

Objective:According to statistics,about 10% to 15% of couples in the world suffer from infertility[1],and infertility caused by purely male factors accounts for about 40%.Studies indicate that genetic factors account for 15% to 30% of male infertility factor [2] and participate in various physiological processes that affect infertility,including hormone homeostasis,spermatogenesis,and sperm quality.Therefore,understanding the genetic basis of spermatogenesis is crucial for the treatment of infertile couples.From the pool of the genes specifically expressed in reproductive tissues,we have succesffuy identified a testis-specific gene,Tbc1d8,and constructed a gene knockout model by Crispr/cas9 technology,resulting in male infertility.Therefore,this topic mainly discusses the role of Tbc1d8 in the process of spermatogenesis and deformation,and discusses its regulatory mechanism.Through the detection of gene expression in various tissues of adult mice including ovary,testis,epididymis,heart,liver,lung,muscle,kidney,small intestine,a series of reproductive specific genes were screened.Among them,Tbc1d8 gene was highly expressed in mouse testis and epididymis.Immunohistochemistry and immunofluorescence showed that the gene was mainly expressed in Leydig cells and spermatogonia.In order to explore the possible role of Tbc1d8 gene in the reproductive system,gene knockout mice were constructed using Crispr/cas9 technology.Depletion of Tbc1d8 resulted in male infertility.Therefore,we have conducted a preliminary discussion on the function of the Tbc1d8 gene in the male reproductive system.The experimental results show that the size of the testis of the mutant male mice is smaller than that of the control group.HE staining shows that the long sperm in the testicular spermatogenic tubules is significantly reduced,and the number of spermatozoa in epididymal is significantly reduced.CASA analysis revealed the loss of sperm motility and forward motion.HE staining of sperm showed a significant increase in the abnormality of sperm head and neck in the mutant mice.Transmission electron microscopy analysis revealed abnormalities in the sperm head.Therefore,Tbc1d8 may play an important role in sperm deformation.Results: Tbc1d8 is highly expressed in the testis and epididymis,and is involved in the process of sperm deformation.Gene mutation results in male infertility.Objective: The oogenesis process for the female mammals contains two important events : the assembly of primordial follicles and the development of follicles.The primordial follicle pool,providing all oocytes available to a female throughout her reproductive life,is established perinatally.The formation of primordial follicle pools is regulated by precise transcription and post-transcriptional levels.In recent years,with the research of non coding RNA function,small molecule non-coding RNA-Micro RNA as a post-transcriptional regulatory factor,involved in the process of primordial follicle assembly,but the contribution of Micro RNA remains unknown.Genomic specific micro RNA-mi R-92 b was screened through literature and database,so this study mainly explored the mechanism of action of mi R-92 b in primordial follicle assembly process.The expression of mi R-92 b in the primordial follicle assembly process was up-regulated by real-time PCR.The in vitro culture system of neonatal mice's ovaries was established,and the assembly of primordial follicles was observed after.knockdown and overexpression were performed by mi R-92 b mimics/inhibitor transfection.The results suggest that inhibition of mi R-92 b can inhibit the cyst breakdown and primordial follicle assembly.The proportion of primordial follicles in the total follicles is significantly reduced(p<0.05),and the expression of follicular development-related genes is reduced.Overexpression of mi R-92 b can accelerate primordial follicles assembly.Through database prediction of mi R-92 b target genes,GO analysis of target genes and signal pathway prediction,mi R-92 b is mainly involved in the regulation of m TOR signaling pathway.The dual luciferase reporter assay demonstrated that mi R-92 b can directly bind to the 3'UTR(3'Untranslated Region)of the target gene TSCl.and can negatively regulate the expression of TSC1 in the ovary.Transfection of TSC1 si RNA in vitro can accelerate primordial follicle assembly.Therefore,we speculate that mi R-92 b regulates p-RPS6 signaling pathway by targeting TSC1 and regulates the process of primordial follicle assembly.Conclusion:Mi R-92 b is highly expressed during primordial follicle assembly and activates p-RPS6 by negatively regulating the target gene TSC1,promoting primordial follicle assembly.