Establishment The Research Of Neonatal Male Rats' Germ Cells Differentiate Into Spermatid-like Cells In Vitro

Purpose: An experiment of the neonatal male rats' germ cells with the method of cell culture in vitro is established.We will study the regulation of the cells differentiation during spermatogenesis by observing the changes of related genes and chromosomes.And the experiment could lay the theoretical foundation for the study of spermatogenesis in vitro.Methods:(1)The testis of 5 days old male rats were taken out and the cells from the testis were prepared and cultured in vitro.The process of proliferation and differentiation were observed by microscope.(2)The markers(DDX4,KI67,PLZF)were detected by immunofluorescence.The chromosomes and markers(STRA8,DMC1,SCP3,?H2AX)were detected by immunofluorescence.The variation trend of them could point out the process of meiosis.The markers(AFAF,GATA4)were also detected by immunofluorescence.(3)qRT-PCR was used to monitor the meiosis markers(STRA8,DMC1,SCP3,KIT)and haploid spermatids marker(ACROSIN)during spermatogenesis.(4)We also used FCM to detect and counted the haploid cells.(5)The structure of spermatids acrosome was observed by PNA stain.Results:(1)The proliferation and morphological changes of the cells in vitro can be observed by microscope in the induced germ cells from 3-20 days.(2)The markers(DDX4,KI67,PLZF)which were detected by immunofluorescence technique at the third day demonstrated that the SSCs were in the proliferation stage of pre-meiosis.Obvious changes of meiosis markers(STRA8,KIT,DMC1,?H2AX,SCP3)were detected by immunofluoresce from 5-10 days.And the immunofluorescence of SCP3,?H2AX and chromosomes demonstrated that the process of synapsis recombination during meiosis.GATA4 and AFAF were detected by immunofluorescence at the 20 th day,the expression of them demonstrated the generation of round spermatid-like cells and the cells had suitable microenvironment.(3)The meiosis markers(STRA8,DMC1,SCP3,KIT)had obvious changes from 5-10 days by qRT-PCR detection.The obvious change of haploid spermatid marker(ACROSIN)was detected at the 15 th day.All above demonstrated that some SSCs were starting the meiosis at the 5th day and there were some haploid spermatids in culture system at the 15 th day.(4)The haploid spermatid-like cells were detected at the 12 th day,then more haploid spermatid-like cells had generated at the 20 th day.(5)We saw the structure of spermatids acrosome by PNA stain clearly at 20 th day.Conclusions:(1)The round haploid spermatid-like cells with normal acrosome structure is first cultured from the neonatal male rats' germ cells by the method of cell culture,and the process of proliferation,differentiation and the events of meiosis are all described.(2)The round haploid spermatid-like cells induced in vitro have shorter time and our experiment is higher efficiency.