Induction Of Germ Cells From Human Induced Pluripotent Stem Cells And The Reproductive Toxicity Of Diethylstilbestrol

Reproductive health is the cornerstone of health.To maintain and promote reproductive health are the pillars,impetus and guarantee for the progress and development of human society.However,many reproductive-related diseases can affect human reproductive health.For example,cryptorchidism is one of the most common congenital birth defects in pediatric urology.Many researches have demonstrated that although the children with cryptorchidism were treated with drug and surgical intervention at early stage,male infertility could still happen when they grow up.The mechanisms of cryptorchidism-related male infertility remain unknown.Embryonic Stem Cells(ESC)and induced Pluripotent Stem Cells(i PSC),by virtue of their abilities of unlimited self-renewal and multi-lineage differentiation potentials,are ideally used for studying the occurrence and development of diseases.Induction of germ cells from ESC/i PSC in vitro could be used to mimic the development process of germ cellsin vivo.It is very helpful to study the mechanisms of various male reproductive-related diseases,drug toxicology and regenerative medicine.Mouse ESC/i PSC could be induced into germ cells efficiently,which contribute to spermatogenesis and to healthy offspring.However,the progress of human ESC/i PSC differentiation into germ cells is relatively slow and most of differentiated cells remained at the early stage.Besides,the induction efficiency and repeatability is low.Therefore,development of in vitro conditions that enable robust differentiation of human i PSC towards late stage germ cells will be necessary.According to the development process of germ cellsin vivo,we constructed the three-step induction method consisting of overexpressing bone morphogenetic protein(BMP4)combined with dihydrotestosterone(DHT)and retinioc acid(RA)to promote the i PSC derived from human somatic cells differentiation into male germ cells.The differentiation potentialswere investigated by morphological changes,real-time PCR,immunofluorescence and manual cell counting method.Result of morphology indicated that the compact i PSC clones began to differentiate in presence of inductionfactors and the differentiated cells were all small round after in vitrodifferentiation for 21 days.Results of real-time PCR and immunofluorescence showed that gene expression of germ cells lineage was notably increased and highly expression of meiosis marker(SYCP3)and spermatocytes markers(TEKT1 and ACROSIN)were detected after in vitro differentiation for 21 days.Result of manual cell counting demonstrated that the percentage of germ cell specific marker(VASA)was about53.52±3.94%after the second-step induction,and the percentage of meiosis and spermatid specific markers SYCP3 and ACROIN was 34.46±2.86%,14.85±1.29% respectively after the third-step induction.Above all,this three-step induction method consisting of overexpressiong BMP combined with DHT and RAcould promote human i PSC differentiation towardsgerm cells lineage efficiently.And a small population of spermatids could be obtained at the last stage.In the second part of this study,we used the germ cells at different developmental stages as in vitro model to detect the effect of diethylstilbestrol(DES)on germ cells development.Our results indicated that high concentration of DES inhibited germ cell proliferation viability significantly,leading to an increase of germ cells death.DES could affect the development and matureof germ cells by influencing the gene expression of early and late stage.Part ?: Induction of germ cells from human induced pluripotent stem cells [Objective] Constructing research models based on human genetic background in vivo and in vitro is meaningful for studying the occurrence and development of male reproductive-related diseases.In our study,we constructed the three-step induction method consisting of overexpressing BMP4 combined with DHT and RA to explore the differentiation potential of human i PSC into male germ cells.[Methods]Firstly,we constructed the Tet-On lentivirus-mediated BMP4 overexpression system.Then there were three steps during the whole induction process:(1)Keep adding Dox for 7days in the first-step;(2)Keep adding DHT for 7days in the second-step;(3)Keep adding RA for 7days in the third-step.The differentiation potential of human i PSC into male germ cells induced by three-step induction method was investigated by morphology,real-time PCR,immunofluorescence and manual cell counting method.[Results](1)Morphological changes indicated that compact i PSC clones began to lose its boundary and the differentiated cells were round or spindle in presence of Dox for 7 days.The differentiated cells were round or spindle-like and there were a lot of cell masses in presence of DHT for 7 days.The differentiated cells were all small-round after in vitro differentiation for 21 days and the overall state of cell differentiation was uniform and synchronous.(2)Results of real-time PCR and immunofluorescence showed that pluripotency genes of OCT4 and NANOG were down-regulated significantly(P<0.05).Primordial germ cells(PGC)specific genes of PRDM1 and STELLA were up-regulated notably after the first-step induction with Dox.For the second-step,pluripotency genes kept down-regulated and PGC specific genes kept up-regulated.The gene expression level of DAZL and VASA which express at the middle-stageof germ cells was increased significantly in the presence of DHT for 7 days.Additionally,gene expression of meiosis marker(SYCP3)and spermatocytes markers(TEKT1 and ACROSIN)began to micro-express.For the third-step,the gene expression of DAZL and VASA began to down-regulated.The expression of SYCP3,TEKET1 and ACROSIN were increased significantly in the third-step(P<0.05).(3)The result of manual cell counting demonstrated that the percentage of VASA was about53.52±3.94% after in vitro differentiation for 14 days,and the percentage of SYCP3 and ACROIN was 34.46±2.86%,14.85±1.29% respectively after in vitro differentiation for 21 days.[Conclusion]The three-step induction method consisting of overexpressing BMP4 combined with DHT and RAcould promote human i PSC differentiation into germ cells lineage efficiently.A small population of spermatid could be obtained at last through this induction method.Part ?: The reproductive toxicityof diethylstilbestrol on germ cells [Objective] Using the germ cells at different stageas in vitro model to detect the effect of diethylstilbestrol(DES)on germ cells development.[Methods]We cultured germ cells of early(day 7),middle(day 14)and late stage(day 21)differentiated from human i PSC by three-step induction method in control group(differentiation medium),solvent control group(0.1%DMSO)and DES concentration gradient of 0.1,1,10 and 50?g/ml for 24 h.Morphological changes were observed by optical microscope.Cell proliferation viability was detected by CCK-8 method and the expression of germ cells specific genes(PRDM1,VASA,SYCP3,TEKT1 and ACORSIN)were detected by real-time PCR.[Results](1)Results indicated that high concentration of DES(50?g/ml)inhibited germ cells proliferation viability significantly,leading to an increase of germ cells death.And the low concentration of DES had no significant impact on germ cells proliferation viability.(2)Compared with the control group,the gene expression in solvent control group had no significant change,and the gene expression of PRDM1,SYCP3,TEKT1 and ACROSIN were decreased significantly(P<0.05)while the expression of VASA had no significant change after being treated with DES in gradient of 0.1,1,10 and 50?g/ml(P>0.05).[Conclusion]High concentration of DES inhibited germ cells proliferation viability significantly,leading to an increase of germ cells death.DES could affect the development and matureof germ cells by influencing the gene expression of early and late stage.