Establishment Of Testicular Organoid And Drug Screening For Male Infertility

Sperms are essential because they can pass on genetic information to their offspring.Spermatogenesis occurs in the testes,which is one of the most complicated differentiation processes in vivo,including proliferation of spermatogonias,meiosis of spermatocytes,and morphological change of spermatids.These processes do not occur autonomously by the germ cells,but require close interaction between germ cells and somatic cells in the testes.It is estimated that 10%-15%of couples undergo reproductive disorders,of which roughly 50%are caused by male infertility.Among them,spermatogenesis disorders result in oligospermia,weak,deformed sperm and azoospermia.Many factors contribute to male infertility,but the mechanisms of infertility remain poorly understood.Therefore,effective diagnosis and treatment options are not available.Despite many advances have been achieved recently,the molecular level of infertility pathogenesis and efficient drug screening are still difficult due to the lack of suitable models of spermatogenesis in vitro.Organoid technology that can mimic the function of in vivo organs,has been rapidly developed.Since organoid greatly facilitates mechanism research and drug screening,it has been widely being used in biomedicine production and drug development.However,there are no testicular organoids that can fully recapitulate the process of in vivo spermatogenesis,and functional sperm cells cannot be obtained.Therefore,we aimed to establish testicular organoid that can generate functional sperms.To begin with,we screened several rounds of three-dimensional materials and culture methods to optimize the system.Ultimately,we established a reconstituted testicular organoid derived from neonatal mouse testicular cells,which is capable of mimicking the process of spermatogenesis in vivo and producing functional sperms by agar culture strategy and combined culture condition.Next,we mapped the atlas and described the traj ectory of spermatogenesis in testicular organoid at the single-cell level for the first time,thus revealing that the testicular organoid could recapitulate the spermatogenesis process in vivo at the molecular level.Next,although functional haploids can be produced by the testicular organoid,its production efficiency is much less than in vivo testes.We performed single-cell transcriptome sequencing combined with bioinformatics analysis to analyze the differences between testicular organoid and in vivo testes.We found a large number of fibroblasts from in vitro organoid when cell types of testicular organoid were identified.Gene ontology analysis further revealed that fibroblasts produced excessive collagen and thus caused fibrosis,which disrupted the homeostasis of microenvironment in the testicular organoid and induced apoptosis of testicular cells.In addition,single-cell transcriptome analysis also revealed defects in the blood-testes barrier in testicular organoid that affect spermatogenesis.Finally,we screened small molecule compounds that targeting fibrotic phenotypes to improve the testicular organoids.As a result,a small molecule named "Pirfenidone(PFD)" could significantly alleviate fibrosis,reducing apoptosis of spermatocyte and therefore enhance the efficiency of haploid production.Then we applied PFD to human testicular tissue culture,which can also reduce collagen produced in the culture process.In summary,we took a lead to establish a testicular organoid which could be capable of recapitulating the entire process of in vivo spermatogenesis and generate functional haploids,providing an important technological platform to investigate the development of germ cells and the molecular pathogenesis of infertility.In addition,small molecule screening targeting fibrosis could not only facilitates research and development of treatments for infertility caused by testicular fibrosis,but also pave the way for the establishment of a human testicular organoid in vitro.In addition,we analyzed the spermatogonial stem cells by using single-cell RNA sequencing and identified a new spermatogonial stem cell gene,Hhex.Knockdown of Hhex in spermatogonial stem cell in and transcriptome comparisons demonstrated that Hhex affects spermatogonial stem cell proliferation by regulating the cell cycle and apoptosis,providing a theoretical basis for the future establishment of the human spermatogonial stem cell.