Study On The Roles And Mechanisms Of MTORC1 Signaling Pathway In Spermatogonial Stem Cells In The Regulation Of Spermatogenesis In Mice

Background and aimsThe mechanistic target of rapamycin(mTOR)is a highly conserved serine/threonine protein kinase.It consists of two distinct complexes:mTORC1 and mTORC2.These complexes have their own function.Tuberous sclerosis complex(TSC)as the significant upstream regulator of mTORC1,inhibits the activation of mTORC1.The activation of mTORC1 can promote protein translation and the generation of ribosome via phosphorylatin-g S6K and 4E-BP1 in the downstream.Mice testis is a significant male reproductive organ.Testis plays an important role in the development of the male reproductive system.Spermatogonial stem cells develop into highly differentiated sperm through mitosis,differentiation and meiosis.More and more evidences indicate that mTORC1 plays an important role in the individual development.However,its indeed function in the development of testis is unclear.Previous results showed that regulatory change in the activation of mTORC1 emerged in the development of testis in C57BL/6 mice.Therefore,mTORC1 associates with the development of testis in mice.The indeed function and mechanism need to be clarified.It possess a guidance to clinical testicular disorder treatment.Methods1?Establishment and propagation of gene knock-out mice.We have successfully created mice with spermatogonia specific deletion of Tscl.We generate male Vasa-Cre-Tsc1flox/flox mice as knock-out mice and male Tsc1flox/flox mice as control in the litter.2?Mice genotype is identified by PCR and agarose gel electrophoresis.The effect of deletion of Tscl was identified by western blot.3?IHC staining and western blot are used to detect p-S6,p-mTOR protein expression in mice testis,thus judging the activation of mTORC1 in the state of specific Tsc1 deletion.4?Analysis on the effect of knock-out mice seminiferous tubule by HE staining.5?Analysis on the situation of knock-out mice testicular cell apoptosis via TUNEL.6?Exploring knock-out mice phenotype underlying mechanisms by detecting the difference in indicator molecules which represent proliferation and differentiation between control mice and knock-out mice.It is performed with Q-PCR,IHC and western blot.7?Sperm counting,detecting the situation of spermatogenesis.8?Establishing mice model of testis destroying by intraperitoneal injection of cadmium chloride,using a dose of 3mg/kg/d and injecting 3 days in a row into 2 months old mice.9?Detection of the expression of mTOR signaling proteins in the cadmium chloride treated mice testis,exploring the mechanism by HE,IHC and western blot.Results1?The testis in knock-out mice is shrinking,spermatogenic cells layer thinningCompared to control mice testis,the testicular weight of knock-out mice declines(58.33±1.53 vs 99.67±2.52,P<0.05),spermatogenic cells layer thinning.2?Significantly increasing number in cell apoptosis,oligospermia emerges in the knock-out miceThe number in the knock-out mice testicular cell apoptosis(NO./mm2)is more than that in control mice(287.33±6.43 vs 151.00±3.61,P<0.05).Epididymal sperm number decreases in the knock-out mice(5.02±0.38 vs 11.10±0.02,P<0.05).3?Abnormal differentiation of spermatogonia emerges in the knock-out mice,thus causing abnormal testicular developmentQ-PCR results show that an obvious decline in Zbtb16(undifferentiated spermatogonial marker)mRNA by about 50%(P<0.05)and increase by 2 fold(P<0.05)in Stra8 and c-Kit(differentiated spermatogonial marker)mRNAs are observed in 3 weeks old knock-out mice.WB shows an obvious decline in Zbtb16 protein and obvious increase in Stra8 protein in 2 months old knock-out mice.IHC shows an obvious increase in Stra8 protein in 2 months old knock-out mice.We point out that abnormal differentiation causes abnormal testicular development in knock-out mice.4?mTORCl activity rises in cadmium chloride treated mice testis,E-Cadherin expression decreasing,testis breaking.mTOR signaling is linked to this phenomenonHE staining shows that the CdCl2 treated mice testis is destroyed.The level of E-Cadherin decreases and the level of p-S6 increases in the CdCl2 treated mice testis.mTORCl activity rises.ConclusionmTORC1 signaling pathway plays an important role in maintaining the spermatogonial stem cell self-renewal.mTORC 1 activation accelerates the differentiation of spermatogonial stem cells.Depletion of spermatogonial stem cells ahead of time induces spermatogenesis disorder,thus resulting in decreased reproductive capacity of male mice.