Study On The Character Of Bovine Male Germline Stem Cells Long-Term Culture In Vitro

Male germ stem cells(mGSCs)settle in testicular seminiferous tubules after sex differentiation and is only one class of adult stem cells which can transfer genetic matter to offspring。Researches on cytology characteristic of mGSCs, methods of separating and purifying of mGSCs, cultural system of mGSCs in vitro, induction of mGSCs into haploid cells or sperm in vitro, gene modification of mGSCs ,transplantation of mGSCs into homogeneity and heterogeneity animals testis have been done extensively。Although new achievements spring out continuously,the study on mGSCs is also at the stage of initiation。Basing on the previous investigations,the aims of presented study are as follow: to develop a simple and effective method of mGSCs separation and purification ; to investigate the characteristic of mGSCs cultured under different incubation system in vitro; to induce mGSCs into sperm-like cells in direction; to clone human c-myc gene and construct。a eukarya expression vector ,contransfection mGSCs with SV40 and hTERT expression vector ,making mGSCs survive and proliferate long-term and immortalize in vitro。We separated bovine mGSCs using two-step enzyme digestion that 0.25% trypsin digestion for 10 min following 0.1% collagenase digestion for 20 min both at 37℃。This method is more beneficial to mGSCs survival。Adhering difference method and percoll density gradient centrifugation can be adopted to purify mGSCs, while we can get mGSCs with more activity and purity by 3 times adhering difference method than the latter。There are two kinds of ball-like cells with different sizes。One kind of cells with small sizes shows dark cytoplasm, adhering after seeding for 1h,projecting after 3h and stretching pseudopod out after 8 to 10h。The other kind of cells with large sizes have a high rate of nuclear/cytoplasm,keeping suspending after seeding 5h and adhering completely after 18～20h。Such cells are all round or ellipse and have a same size。mGSCs started to proliferate after seeding for 2d,appearing 2-cells or 4-cells mass。mGSCs formed small colonies after seeding for 5d,showing positive for AKP staining。We also studied the effects of serum concentration , Sertoli cells ,mGSCs seeding density,SCF,LIF and GDNF on proliferation of mGSCs in vitro,the identification of mGSCs was also conducted。Experiment 1: add 0%,2.5%,5% and 10% fetal bovine serum(FBS), respectively, to the basal culture medium(Dulbecco minimal essential medium (DMEM) with high glucose)。The results showed that DMEM with 2.5% FBS was mostly proper for the proliferation of bovine mGSCs in vitro。Experiment 2: Sertoli cells were seeded at six densities as feeder layer。We found that mGSCs proliferated significantly(p<0.05) when Sertoli cells were seeded at density of 1.0×106/mL。Experiment 3: mGSCs were seeded at densities of 1.0×104/mL,5.0×104/mL,1.0×105/mL and 2.0×105/mL on the feeder layer。The result showed that the group seeding 2.0×105 cells/mL formed colony first and have the highest relative colony forming rate (p<0.05)。Experiment 4: adding SCF, LIF or GDNF to the medium could increase the number of SSCs。However, only the concentrations of SCF,LIF and GDNF 20,80,10μg/L, respectively, could significantly increase the number of SSCs compared to the control group(P<0.05)。Alkaline phosphatase(AP) staining , immunohistochemical staining and RT-PCR were conducted to identify bovine mGSCs。The results showed that AP staining was weakly positive ; Integrin .β1 and C-kit staining were both positive。The sequences of Gfrα1 and c-kit were also obtained by RT-PCR, the sizes(120 bp and 280 bp) were identical to the expected sizes。Therefore , DMEM with 2.5% FBS ,adding 20μg/L SCF,80 ng/mL GDNF and 10μg/L LIF ,respectively, was appropriate for bovine SSCs culture in vitro。We cloned 3.6 kb c-myc by PCR, the fragment containing the second and the third extron of c-myc and coding a protein of 439 amino acids。We then linked c-myc and hTERT into the pEGFP vector and constructed two vectors : pEMY and pETE。mGSCs were transfected by pCI- neo-hTERT+pSV3-neo,pEMY+ pCI- neo-hTERT,pCI- neo-hTERT and pEMY。After screening , four putative cell lines were obtained and named GTS1,GMT5,GT3,GM7。The four cell lines have 35,28,29 and 28 passages ,respectively。The rates of contransfections by pCI- neo-hTERT+pSV3-neo,pEMY+ pCI- neo-hTERT were higher than that by single vector。The cell lines- GTS1,GMT5,GT3,GM7- all expressed GFR -1, Oct-4, c-kit and Integrinβ1。We induced mGSCs into sperm-like cells by RA, testes tissue juice of adult bovine and low temperature。The rate of haploid is 19.3% ,examined by flowcytometer。These sperm-like cells could activated bovine oocytes by intracytoplasmic sperm injection (ICSI), the rate of blastula up to 20.36%。...