Study On Differentiation Of Embryonic Stem Cells Derived From Embryos Donated By Robertsonian Translocation-carried Male Infertility Couples Into Germ Cells

About 10% to 15% couples of childbearing age have infertility problems,of which male factors account for 50%.More than 15% male factor infertility are related to genetic disorders.However,the study on the potential pathomechanisms was confined by appropriate materials and animal model,as well as ethics.Embryonic stem cell(ESC)can be differentiated into all the cell types of the three germ layers,including sperm cells and even active sperm.The process of human ESC(h ESC)differentiating into sperm in vitro reproduces the generation of spermatozoa in the body.In the same way,if the in vitro differentiation of ESC is abnormal,it may indicate the disorder of spermatogenesis in the body.At present,ESC has not been induced to the well-functional sperm like mouse ESC.However,h ESC has become an ideal model cell to study the mechanism of human spermatogenesis.In this study,the h ESC system was successfully established by 2 infertile couples.One of the husbands,carried with chromosome Roche translocation,has severe oligozoospermia.And the other one has severe oligozoospermia,carried with chromosomal balanced translocation.The indentified h ESCs were in vitro differentiated into spermatozoa,and the expression levels of some sperm cell markers were analyzed in the process of differentiation.The key genes and mechanism of spermatogenesis were preliminarily explored.The specific h ESCs will provide an reference for us to evaluat the health and genetic risk of the assisted reproductive technology(ART)babies from fathers suffered from severe oligozoospermia andasthenozoospermia.Material and Methods1.Establishment of embryonic stem cell lines: The embryos donated by two couples with severe oligozoospermia and asthenozoospermia carried with chromosome Roche translocation and chromosome balanced translocateion,donated during their ART treatment with the informed consent.The ESC was established by separating the cell mass of blastocyst,and then underwent the in vitro culture,generation and amplification.2.The identification of two cell lines: the pluripotent activity of h ESC was detected by immunofluorescence,embryoid formation,formation of teratoma in vivo and real-time quantitative polymerase chain reaction(RT-q PCR).The karyotype of chromosomes was tested by gene sequencing.3.The in vitro differentiation of h ESCs into spermatozoa and analysis: The differentiation of these h ESCs was in vitro induced by adding retinoic acid(RA)into the ES medium.The differentiation process of the germ cells and the expressions of related genes were then analyzed through the morphology of embryoid bodies(EBs),immunofluorescence and RT-q PCR.A routine h ESC from our laboratory,with the normal genetic background,was used as control.Results1.Establishment and identification of embryonic stem cells1.1 Establishment and identification of CCRM16 The h ESC line derived from an embryo donated by severe oligoasthenospermia patient carried with chromosome Roche translocation,named as CCRM16,was successfully established.The chromosomal G band analysis showed that thekaryotype of the CCRM16 was 46,XY,+14,rob(13;14)(q10;q10).CCRM16 has a typical clonal growth,with obvious boundaries,large nuclei,clear nucleolus and high nuclear ratio.CCRM16 showed typical h ESC characteristics: expressing pluripotency makers of NANOG,OCT4,SSEA4,and TRA-1-181,forming embryoid bodies(EBs),and differentiating into the three germ-layer tissues in vitro and in vivo.1.2 Establishment and identification of CCRM27 Another h ESC line,CCRM27,derived from an embryo donated by severe oligoasthenospermia patient carried with chromosome balanced translocateion,was established.The chromosomal G band analysis showed that the karyotype of the CCRM27 was 46,XY.The karyotype of the embryo was inconsistent with the h ESC.CCRM27 has a typical clonal growth,with obvious boundaries,large nuclei,clear nucleolus and high nuclear ratio.CCRM27 showed typical h ESC characteristics: expressing pluripotency makers of NANOG,OCT4,SSEA4,and TRA-1-181,forming embryoid bodies(EBs),and differentiating into the three germ-layer tissues in vitro and in vivo.2.Differentiation of embryonic stem cells into germ cell2.1 Differentiation of CCRM16 into germ cell A normal h ESC,CCRM23,was used as control.CCRM16 was induced and differentiated into the germ cell,spermatozoa,using 2?M retinoic acid(RA)in ES medium.The expressions of DAZL as primordial germ cell(PGC)marker and SCP3 and VASA as two meiosis markersin the differentiated CCRM16 were significantly decreased when compared with those in the CCRM23 underwent the same treatment.When CCRM27 differentiated into germ cells by 2?M RA,the expressions of DAZL,SCP3 and VASA were decreased compared with CCRM23.Interestingly,the expression levels of STRA8,SMC1 B and TEX11,which were directly regulated byDAZL,were decreased significantly compared with CCRM23.2.2 Differentiation of CCRM27 into germ cell CCRM23 was also used as control in this experiment.When CCRM27 was induced and differentiated into germ cells by 2?M RA,the expressions of DAZL as a PGC marker and SCP3 and VASA as two meiosis markers were significantly decreased when compared with the CCRM23(46,XY)underwent the same treatment.The expression levels of STRA8,SMC1 B and TEX11,which were directly regulated by DAZL,decreased significantly compared with CCRM23.The general genome DNA sequencing did not find over 4M DNA deletion on any of chromosomes.Conclusion 1.Two h ESCs with the severe oligoasthenospermia background,CCRM16 and CCRM27,have been successfully established in this study.They have typical characteristics of embryonic stem cells.2.The differentiation into early sperm cells was abnormal.The phenotypes of the differentiated CCRM16 and CCRM27 are consistent with the clinical findings.3.The in vitro differentiation of CCRM16 and CCRM27 into sperm as a research model can be used to explore the key genes related to spermatogenesis and the mechanism of spermatogenesis.Meanwhile,this model provide us an experimental basis for evaluating the health risk and genetic risk of the ART babies derived from those fathers with severe asthenozoospermia.