| Spermatogenic stem cells are located on the basement membrane of the convoluted spermatozoa of testis and are mainly involved in spermatogenesis.It maintains the stability of the reproductive stem cell bank through self-renewal or self-differentiation into a certain number of spermatozoa and eventually develops into sperm,ensuring the normal reproductive ability of male animals.SSCs is the only male animals can pass genetic information to offspring of stem cells,have very strong plasticity,not only can the culture building or establish its long-term training system,will greatly promote the spermatogonial stem cell transplantation,the practical application of transgenic technology,and will provide the development of in vitro cell differentiation with good material.Researchers to foster transgenic animals are the main methods of early embryos implanted or cells in the animal design of genes,but birds for egg-laying animals,birds of SSCs in the production of transgenic chicken and save endangered species in the genetic resources has a broad application prospect,and establish a can make effective breeder poultry SSCs and represented the in vitro culture system is a precursor of the application conditions.This research is divided into five parts:Experiment 1: Expression characteristics of undifferentiated specific markers of pluripotency in spermatogonial stem cells from different age chicken testis tissues in Huaixiang.Five chickens from each of 6 different ages(8d,21 d,31d,45 d,58d and 79d)were selected,and one of the testis tissues from Huaixiang was used for histological sections and immunofluorescence staining of white slices.The relative expression levels of Gfra1,CD90 and OCT4 m RNA in testicular tissues of 4 huaixiang chickens were determined by qPCR.The results show that:(1)With ontogenesis,SSCs gradually migrated from the middle part of spermatogenic tubules to the basement membrane.The number of SSCs positive cells decreased as the day age of chickens in Huaixiang increased and the cells in the testis gradually differentiated to the late cells.(2)QPCR detection different day age of testicular tissue spermatagonial stem cell pluripotency undifferentiated specific marker gene expression results show that with the increase of homesickness chicken day age,testicular tissue specificity of marker genes Gfra1,CD90,OCT4 m RNA expression gradually reduce,SSCs positive cells number less,adult chicken testicular tissue in SSCs will still exist,but content reduce obviously,SSCs in vitro separation efficiency decreases.The younger the chicks were,the more SSCs positive cells they had,and 21 d age was selected as the subsequent SSCs separation age.(3)Immunofluorescence staining showed positive Gfra1 and CD90 in the paraffin sections of chicken testis from Huaixiang.Experiment 2: The effect of different age on the efficiency of spermatogenic stem cells isolated from testicular tissue(1)Three two-step enzyme digestion method(1 mg/m L Ⅳ collagenase type and 30 mu g/m L Dnase enzyme trypsin + 0.02 + 0.125 g g EDTA)processing 6homesickness chicken testicular tissue in different periods,the age of 21 d the total number of cells they 3.37 x 106,compared with 45 d 8 d,31 d,total number of cells was reduced by 29.37%,32.64% and 24.63% respectively(P < 0.01);The total number of day-old cells on 58 d and 79 d increased by 47.99% and 48.63%respectively(P<0.01).There was no significant difference in survival rate among8 d,21d,31 d and 45 d aged cells(P>0.05).Compared with 58 d aged cells,survival rate decreased by 7.38%,8.86%,6.23% and 7.06%,respectively(P<0.05).The survival rate of 79d-aged cells decreased by 9.09%,10.54%,7.97%,and 8.78%,respectively(P<0.05).This indicated that the older the chicken age in Huaixiang was,the larger the testicular tissue was,and the number of cells isolated increased.However,the time required for complete digestion of testicular tissue became longer,resulting in the decrease of cell survival rate due to overdigestion.The optimal age of spermatogonial stem cells was 21 days.Experiment 3: The effects of different digestive enzyme combinations and different purification methods on the yield and viability of spermatogonial stem cells21 days of age and homesickness chicken eight rooster,digestive enzymes were randomly divided into combination group 1(1 mg/m L Ⅳ collagenase type and 30 mu g/m L Dnase enzyme trypsin + 0.04 + 0.25 g g EDTA),combination of digestive enzymes,2 groups(1 mg/m L Ⅳ collagenase type and 30 mu g/m L Dnase enzyme trypsin + 0.02 + 0.125 g g EDTA),each group of four.The cell count plate was used to count the cell rate after digestible testicular tissue by digestive enzymes in the two groups,and the cell viability was calculated by trypan blue staining.After the separation,the SSCs were divided into three parts to compare the efficiency of SSCs before and after purification by differential wall sticking method,Percoll density gradient centrifugation method and phytophric magnetic bead separation method.The results show that:(1)The average yield of digestive enzyme combination 1 and 2 per gram of testicular cells was(4.25 ± 0.9)× 106 and(3.22 ± 0.89)× 106(P>0.05),respectively.Digestive enzymes combination method cells at a rate of 1(83 + 2.13)%,compared with digestive enzymes combination method of 2 cells rate increased by 16.18%(P < 0.01),the digestive enzyme digest testis cell activity obtained higher portfolio 2,combination of digestive enzymes were used for separation of chicken SSCs,showed that low concentration composite enzyme(2)digestive enzyme concentration is more suitable for separation of testicular tissue,more conducive to the cultivation of the subsequent cells.(2)The single-cell suspension was obtained by digestion of digestive enzyme combination 2.The single-cell suspension was purified by three different purification methods(differential adherent method,Percoll density gradient centrifugation method,and immunomagnetic bead separation method),and the purification efficiency was(81.19 ± 0.16)%,(77.39 ± 0.57)%,and(49.53 ±0.72)%,respectively.The purification efficiency of differential wall sticking method and density gradient centrifugation method increased by 63.92% and56.24%,respectively,compared with the immunomagnetic bead method(P <0.01).This indicates that differential adherence method can be more enriched into SSCs.In the case of no difference in purification efficiency,differential adhesion method is more convenient and faster than density gradient centrifugation.In general,the differential adherent method is more suitable for the purification of cells in our laboratory.Experiment 4: The effect of culture medium with different concentrations of serum on the proliferation of primary spermatogonial stem cells in vitro2 digestive testicular tissue cells in low concentration digestive enzyme combinations differential sticking wall after collecting the cell suspension,testing different concentrations of serum(2%,5%,7%,10% FBS)four nutrient solution formula of chicken essence of the original number of stem cell clone,AKP positive affect,cellular immune fluorescence detection Gfra1 SSCs markers and OCT4,qPCR detection Gfra1 SSCs key transcription factors and the influence of the expression level of OCT4.The results show that:(1)The 2%FBS group could significantly promote the proliferation of spermatogonial stem cells,and the mean colony number of spermatogonial stem cells in the same time was significantly different from that of other experimental groups(P<0.01).Low concentration serum culture was necessary for the proliferation of spermatogonial stem cells in vitro.(2)AKP staining and immunofluorescence staining of Gfra1 and CD90 in the clusters of botrytis cinerea cells showed positive results,indicating that Gfra1 and CD90 were specific marker genes of SSCs,corresponding to the expression of Gfra1 and CD90 in testicular tissue.(3)The expression levels of key SSCs transcription factors Gfra1,CD90 and OCT4 m RNA were significantly different from those in the supporting cells(P<0.01),indicating that only SSCs expressed Gfra1,CD90 and OCT4 in the testis.Experiment 5: The effect of cytokines(GDNF,m LIF and b FGF)on the proliferation of primary spermatogenic stem cells in vitroCCK8 method was used to detect the OD values of single factor concentration and combination factor concentration.The single-factor experiment was set up in five groups,namely the control group and the single-factor group with different concentrations,with 5 replicates in each group.The combination factor was set in 20groups(see experimental grouping)with 5 replicates per group.The results show that:(1)When the three cytokines were added separately,the optimal concentrations of GDNF,b FGF and m LIF were 20ng/ml,30ng/ml and 5ng/ml,respectively.When the three cytokines were added together,the proliferation of SSCs was promoted at low dose and inhibited at high dose.It was concluded that the optimal dose of GDNF,m LIF and b FGF was 5-10ng/ml,5-10ng/ml and 10-20ng/ml,respectively.This test showed that the method of digestive enzyme combination 2(low concentration group)differential adherence to the testis tissue of the chicken roosters at 21 days of age was the most suitable for the separation and purification of SSCs.The formula of 89%DMEM+1% double resistance +2%FBS+8%KSR+5.5×10-5mol/L 2-thiol ethanol +1mmol/ L pyruvate l-glutamine +0.1mmol/ L non-essential amino acid +10ng/ml GDNF+10ng/ml m LIF+20ng/ml b FGF was beneficial to the proliferation of SSCs. |
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