Germ Cell Meiosis In Vitro To Obtain Functional Spermatids

In recent years,according to the statistics of WHO,infertility has plagued about 15% childbearing couples in the worldwide,among them,male infertility accounted for about half of the total number.So far infertility can only be treated by assisted reproductive technology.However,due to the technical limitations,it requires both spouses to provide health eggs and sperms by assisted reproductive techniques,including in vitro fertilization(IVF)and intracytoplasmic sperm injection(ICSI).To the infertile male caused by the lack of functional sperms,they have to accept the sperms from sperm bank,so they can't get babies derived from their own genomes.Therefore,through the methods of cell culture of pluripotent embryonic stem cells and tissue culture of testicular tissue in vitro after germ cell differentiation and meiosis to obtain the function sperms can provide hope for azoospermia patients.In this study,we focused on the functional sperms through artificial culture in vitro.We mainly used the mouse embryonic stem cells and neonatal mouse testes and human adult testis tissue by cell and tissue engineering to make them complete meiosis,and ultimately produce fertile sperms.Detail summaries were as below.1.The developmental function of spermatid like cells derived from ESCs meiosis in vitro.To mimic the germ cell development in embryonic mice,we first differentiated the embryonic stem cells into ectoderm like cells(Epiblast like cells,EpiLCs),then stimulated the EpiLCs to PGCLCs by the key factors of PGC induction.Subsequently,the PGCLCs were purified by PGC specific Blimp1-mVenus and Stella-ECFP reporter system or PGC specific surface marker SSEA1 and Intergrin-?3.We mixed the purified PGCLCs with testicular cells from sertoli-only mice,the PGCLCs could initiated and finished meiosis in vitro with the effect of several cytokines and hormones.Thereafter,we could use flow cytometry to get haploid cells in the mixture of the testis cells of azoospermia mice and PGCLCs after the culture of two weeks in vitro.These haploid cells were about 8~9 ?m in diameter,small and round,with a clear area of chromatin mass.The results of immunofluorescence staining showed that these cells expressed the germ cell marker DDX4 and showed the presence of sperm specific acrosomal granules or acrosomal cap by PNA.The methylation pattern of H19 was completely established,and the maternal imprinting of Snrpn was almost completely open,indicating that its epigenetic characteristics had been established.We analyzed the expression profiles of these haploid cells and found that these cells were closest to round spermatids from testis.Since the haploid cells derived from in vitro meiosis were similar to the round spermatids in the seminiferous tubules in many respects,we named the haploid cells as spermatid like cells(SLCs).Subsequently,we performed round spermatid injection with SLCs to verify their developmental potential,which is also the "gold standard" of gametes.Finally,we could obtain 9 health and fertile offspring from 379 reconstituted embryos derived from two different embryonic stem cells.So far,this is the first one to achieve functional male gametes from mouse pluripotent stem cells meiosis in vitro.It provides a new idea and a good basis for the study of stem cell differentiation in the treatment of male infertility.And we believe that it could bring the gospel for infertility in the future through continuous efforts of scientists by the application in other species or human.2.To obtain the functional gametes from mouse testis tissue in vitro efficiently.We cultured the testicular seminiferous tubules in condition of gas-liquid combined environment using agar as support.And then through the addition of previously reported cytokines and hormones which can promote meiosis and spermatogenesis,we tried to screen factors which can promote the development of meiosis and spermatogenesis in tissue culture conditions.In seven added factors,through the detection of the haploid content in seminiferous tubules,we found that testosterone(T)and bovin pituitary extract(BPE)could generate haploid cells more efficiently than the other groups.In a more detailed detection in different time points,we found that BPE was able to stimulate the seminiferous tubules in an earlier and more efficient manner.Under these conditions,the proportion of haploid cells in the BPE group was about 12%,and the haploid cells had the typical size and chromatin distribution as round spermatid in vivo.Subsequently,in order to verify the normal gamete developmental potential of the haploid cells obtained by tissue culture,we carried out ROSI.The control group and BPE group were injected with 66 and 44 oocytes respectively,90.9% and 95.5% embryos can develop to 2-cell stage.Finally,2 healthy offspring were born in both two group after the embryo transplantation into pseudopregnant mice.In this study,we first found that pituitary extract has an important role in promoting the spermatogenesis of testis tissue culture in vitro.Compared with the traditional two-dimensional cell culture,tissue culture can better preserve the signal transduction and interaction between cells.Therefore,testicular tissue culture can simulate the process of meiosis and spermatogenesis more realistic.With the help of this culture system,we study the process of spermatogenesis more detail,and it provided more reference for the application of this system to the human testis tissue culture.In summary,this study provides new ideas for the study of artificial sperms,and it provides a theoretical basis for the clinical treatment of male infertility.