The Mechanism Of DMRT1 On Regulating Differentiation And Self-renewal Of Goat Male Germline Stem Cells

Male germ stem cells(mGSCs),also called as spermatogonial stem cells(SSCs),are adult stem cells located on the basement membrane of testicular seminiferous tubules,which can not only maintain the stability of population through self-renewal,but also differentiate into sperm cells.mGSCs can be used as a model for the study of spermatogenesis,meiosis regulation and cell reprogramming.The protein encoded by Double sex and mab-3 related transcription factor 1 gene contains a double sex/mab-3 domain,which is the most conservative domain in both vertebrates and invertebrates,and DM domain involved in individual sex determination.In addition,DMRT1 participates in the repair of mGSCs injury,inhibits testicular tumors,maintains the balance between mitosis and meiosis of mGSCs,and affects the pluripotency of spermatogonial stem cells.Therefore the research on mechanism of DMRT1 in mGSCs can provide theoretical guidance for improving animal breeding,animal models of disease and species protection.In this study,we first explored the effects of DMRT1 on the differentiation and self-renewal of mGSCs in dairy goats and the functions of DMRT1 in reproductive immunity.The experimental results as follows:1.DMRT1 gene widely exists in mammals,the sequences of DMRT1 genes in different species are highly conservative.The DMRT1 gene sequence alignment of 6 mammals was 86.42 % in consistent.For DM domain analysis,we found that the amino acid sequences of DM domains in different species are basically in same,indicating that DM domains are highly conserved among different species as well.In order to investigate the expression of DMRT1 in different tissues of goat,immunofluorescence staining showed that DMRT1 was specifically expressed in the testis.Immunofluorescence staining and quantitative PCR further showed that the expression of DMRT1 was very low in 1-month-old goat testis.The expression of DMRT1 was significantly up-regulated after the initiation of meiosis at 3-months-old goat testis,and it tended to be stable at maturity.Secondly,revealed by immunofluorescence co-staining that DMRT1 was mainly localized in undifferentiated and differentiating male germ stem cells,but it was not expressed in spermatocytes that initiated meiosis.According to these results,it was speculated that DMRT1 mainly plays a role in male germ stem cells and takes an essential place in the differentiation and proliferation of mGSCs.2.FUW-TetO-Dmrt1 lentivirus vector supplement with rtTA helper vector were transfected into mGSCs and immortalized mGSCs-I-SB cell line,and the optimal Dox concentration was screened.EDU,dual-luciferase assay,immunofluorescence staining,Western blotting and quantitative PCR further revealed the role of DMRT1 in development of goat mGSCs.The proliferation and differentiation level of mGSCs were significantly increased under 1?g/mL Dox induction,and the corresponding markers of meiosis were downregulated.We further verified the meiosis of RA initiating mGSCs,however,overexpression of DMRT1 inhibits meiosis,while up-regulation of proliferation marker PCNA and differentiation marker c-KIT of mGSCs.On the one hand,DMRT1 promoted the proliferation and differentiation of male germline stem cells.On the other hand,it inhibited the abnormal initiation of meiosis caused by RA,thus maintaining the balance between mitosis and meiosis of spermatogonia.3.DMRT1 played an important role in inhibiting reproductive immunity.DMRT1 can reduce inflammation by blocking TLRs signaling pathway.Transcriptional sequencing and immunohistochemical results show that TLR4 and NF-?B are activated in DMRT1-interfered mGSCs.ELISA and Western blotting assay demonstrated the expression of pro-inflammatory factors TNF? and IL-6 in mGSCs were up-regulated.Immunocoprecipitation and dual-luciferase assay further revealed that DMRT1 recruited PLZF protein,which together inhibited the transcription of TLR4 and NF-?B and promoted the replenishment of mGSCs.Meanwhile,TUNEL staining and flow cytometry showed that DMRT1 inhibited apoptosis of mGSCs induced by inflammation.These results demonstrated the important role of DMRT1 in promoting self-renewal of spermatogonial stem cells and maintaining homeostasis of testicular environment,which provided a new way to treat reproductive defects.4.The testis of mice treated with bulsufan showed severe reproductive damage.Injection of DMRT1 lentivirus into seminiferous tubules significantly restored the number of mGSCs and maintained spermatogenesis.The results of immunofluorescence staining and ELISA showed that DMRT1 promoted the expression of PCNA,VASA,PLZF and CSF1 R in male germline stem cells.At the same time,the expression of cytokines GDNF and CSF1 were up-regulated,which proved that DMRT1 is essential for the reconstruction of testicular internal environment.Based on the detection of bimolecular fluorescence complementarity(BiFC)and immunoprecipitation,the interaction between DMRT1 and PLZF was mainly depended on the interaction of the BTB domain and DMRT1.Furthermore,DMRT1 and PLZF play a synergistic role in regulating the downstream pathway of self-renewal in mGSCs.In addition,DMRT1 and PLZF as anti-inflammatory factors effectively inhibited the occurrence of inflammation in seminiferous tubules and establish environment for the repair of germ cell damage.In conclusion,we first explored the expression pattern of DMRT1 in mGSCs,uncovered the effect of DMRT1 in differentiation and self-renewal of mGSCs in goats,and revealed the important function of DMRT1 in reproductive immunity.Gene-modified vectors were transfected into dairy goat male germ stem cells.Screening of specific target gene of DMRT1 and its interaction in vivo and in vitro,which provide not only a reliable scientific basis for studying the mechanism of self-renewal and spermatogenesis of goat mGSCs but also a new scheme for inheritance,breeding and prevention of reproductive infertility in high-quality livestock.