Study On The Functional And Molecular Mechanisms Of Tumor Suppressor Gene P53 In Maintenance And Regulation Of Spermatogonial Stem Cells Fate

Spermatogonial stem cells（SSCs）are germline stem cells with the abilities of self-renewal and differentiation to sperm in the testis of male animals.The number of spermatogonial stem cells is limited,only accounting for 0.02-0.03%of all germ cells.Maintenance of number and stability of spermatogonial stem cells is pivotal for male fertility.However,many factors,such as environmental toxins,stress,cytokines,etc.,can affect the fate of spermatogonial stem cells and interfere in spermatogenesis.Study found that stress and autophagy induced by chemotherapy drug busulfan or abnormal cytokine levels,are important factors that affect the fate of spermatogonial stem cells,causing the damage of spermatogonial stem cell maintenance and self-renewal.However,the exact molecular mechanism remains unclear.In this study,we investigated relevant regulatory molecules and signaling pathways that regulate the fate of spermatogonial stem cells in three different directions.Chapter 1:Functional and molecular mechanisms of autophagy in spermatogonial stem cells apoptosis induced by busulfan.In recent years,more and more evidences have revealed that autophagy has a protective effect on the maintenance of stem cells under exogenous stress.Moreover,the chemotherapeutic drug busulfan can cause stress and damage to spermatogonial stem cells associated with significant change in autophagy level.In order to better understand the role of autophagy in cell apoptosis,we studied the potential function of autophagy under the stress of busulfan.And we found that busulfan treatment could induce the formation of autophagic vesicles and autophagosomes in mouse spermatogonial stem cells,and inhibit the autophagosome degradation.Subsequently,Western blotting and immunofluorescence showed that autophagy regulated the maintenance and survival of spermatogonial stem cells in a dose-dependent manner.Low doses of autophagy protected spermatogonial stem cells from busulfan injury,while high doses of autophagy exacerbated the damage of spermatogonial stem cells.In addition,we also demonstrated that Mtor is an important factor regulating autophagy in spermatogonial stem cells.The mechanism of Mtor regulating autophagy in spermatogonial stem cells was very complex:（1）MTOR was phosphorylated by AKT to activate its target genes,P70s6k,resulting in the inhibition of autophagy during short-term busulfan treatment.（2）Mtor and p53 can co-regulate the autophagy of spermatogonial stem cells and affect the fate of the cells.This chapter research provided a new mechanism for autophagy to regulate spermatogonial stem cells.Moderate amount of autophagy could effectively protect spermatogonial stem cells from the stress of chemotherapy,which may provide important hints for clinical protection of male fertility.Chapter 2:Study on the molecular mechanism of p53 in spermatogonial stem cells reprogramming using ATAC-seq and RNA-seq.It was found that unipotent mouse spermatogonial stem cells spontaneously transformed into ES-like pluripotent stem cells under in vitro culture conditions,and the molecular and cellular characteristics of these cells are similar to embryonic stem cells.In addition,studies have found that the spontaneous conversion rate of p53 deficient spermatogonial stem cells was significantly increased in vitro culture,and it has been confirmed that p53 can regulate the expression of some pluripotent-related genes.However,the molecular mechanism of pluripotent transformation of these cells remained unclear.To reveal the transformation mechanism,we first confirmed that there was no significant difference in morphological characteristics,growth characteristics and molecular marker expression between p53^+/+and p53^-/-spermatogonial stem cells.Then,p53^+/+and p53^-/-spermatogonial stem cells were cultured in vitro for a long time and transformed into ES-like cells by pluripotency induction.It was confirmed that the morphological characteristics,growth characteristics and pluripotent expression profiles of p53^+/+and p53^-/-SSCs derived ES-like cells were very similar to embryonic stem cells,and teratoma with three germ layers formed after subcutaneous injection of these cells in nude mice.Based on ATAC-seq and RNA-seq analysis results,we screened out the differential chromatin open regions and differentially expressed genes between p53^+/+and p53^-/-spermatogonial stem cells,and explored the molecular mechanism that p53 deletion induced pluripotent transformation of spermatogonial stem cells.According to the analysis results of ATAC-seq and RNA-seq,it was predicted that Smad3 may be an important transcription factor that induced pluripotency of p53 deficient spermatogonial stem cells.Subsequently,by adding SMAD3 activator（Alantolactone）or inhibitor（SIS3）to spermatogonial stem cells and interference and overexpression of SMAD3 was treated spermatogonial stem cells,The potential function and molecular signaling pathways mechanism of Smad3 in p53 deficient spermatogonial stem cells reprogramming was confirmed.Research results in this chapter are helpful to understand the biological characteristics and rules of unipotency and pluripotency germline cells,as well as the function and molecular mechanism of p53 in maintaining stem cells genome stability,maintaining the fate of cells and regulating apoptosis.This research had certain guiding significance in revealing the fate regulation of spermatogonial stem cells and its application in clinical medicine,especially in the study of male gonad tumor.Chapter 3:Establish a new system of spontaneous reprogramming of SSCs and explore its molecular mechanism.Although spermatogonial stem cells can only differentiate into spermatozoa in the testicular microenvironment,spermatogonial stem cells cultured in vitro have the potential to be spontaneously induced into pluripotency.There are many disadvantages in somatic cell reprogramming methods,such as the long transformation time,the risk of oncogene or virus and the low efficiency.Therefore,the spontaneous reprogramming of spermatogonial stem cells to obtain pluripotent stem cells could be a good option.Although spontaneous reprogramming of spermatogonial stem cells was reported in2004,the efficiency of the transformation method was extremely low（the repetition rate was only 19%,and 1.5x10⁷ cells could generate 1 transformed cell）,and the molecular mechanism of spontaneous reprogramming remained unclear.We established an efficient in vitro spontaneous reprogramming system of spermatogonial stem cells by processing and improving the spermatogonial stem cell culture system and adjusting the addition of growth factor LIF/EGF and serum,which can stably obtain pluripotent stem cells from spermatogonial stem cells.And we called these cells GSPCs.There was no significant difference in morphology,expression of specific molecular markers and methylation expression level between GSPCs and pluripotent stem cells according to previous reports.GSPCs were injected subcutaneously into nude mice to form teratoma with three layers,and the cells were injected into blastocysts to form blastocyst chimerism.These results proved that GSPCs are pluripotent stem cells.In our conversion system,GSPCs forms as early as10 passages,remarkably less than previous reported system requiring 30 passages,and the repeatability rate of each independent assays is more than 90%.Subsequently,SSCs,intermediate stage-cells（In state）and stably transformed stage-cells（GSPCs）were harvested for RNA-sequencing,to explore the molecular mechanism of SSCs spontaneous reprogramming.These results suggest that TGF-β-SMAD signaling pathway plays an important role in SSCs reprogramming.In summary,the regulation of SSCs fate is pivotal for male reproduction and cloning technology.We used male mice spermatogonial stem cells as model animals to deeply study the protective effect of autophagy on fertility and the molecular mechanism of spermatogonial stem cells’spontaneous transformation into pluripotent state.These results provide theoretical support for further research in revealing the molecular mechanism of spermatogonial stem cell maintenance and pluripotency induction in vitro,provide valuable reference for the application of fertility protection in clinical chemotherapy,a basis for clinical treatment and diagnosis;and new ideas for male reproduction maintenance and gene cloning in animal production.