| Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, can produce a lot of sperm and maintain male fertility by proliferation and differentiation in animal life. However, SSCs are very few in animal testes, and proliferated by establishment of an effective culture system in vitro, then sufficient amount of SSCs were obtained. After SSCs was regulated genetically, they were transplanted to the donor animal or differentiate into sperm cells or complete sperm in vitro. Establishment of SSCs culture and differentiation system in vitro has broad application prospects for cloning animals, transgenic animal production and gene therapy of some human genetic diseases in the field of medicine and livestock production. Goat is an affordable and pregnancy short animal, it can be used to produce a variety of genetically modified recombinant proteins and valuable products. Therefore, study on effect of regulation factors on the proliferation and differentiation of goats SSCs, this will establish transgenic goats models. The production of recombinant proteins and a variety of genetically valuable products is more important significance in biomedical research, agricultural products and biopharmaceutical industry. In order to gradually establish an effective goat SSCs culture system in vitro, the main topic of research carried in the following:(1) Suitable age goat used as a isolated SSCs donor was selected; appropriate digestion method was investigate for isolated testicular cell suspension; suitable purification method was also selected for purifying goat SSCs. Methods of separation and purification for goat SSCs were established; (2) The aim of this study was to compare to effect and mechanism of three feeder layers on growth of caprine SSCs in-vitro; (3) Effect of adding Vc in cell culture on the growth of caprine SSCs was investigated; (4) Effects of different graft site and HGF on the ability of seminiferous tubules reconstructed and differentiation of cells were studied after transplantation; (5) Effects of four different media on goat SSCs differentiation were studied in vitro. The results are as follow:(1) Total testicular cells were the most from 5-month goat, while were minimal from 1-month goat. Thy-1 positive cells in testicular cell suspension from 1-month goat were the most. The number of caprine SSC colonies developed from 1-month goat was significantly elevated compared with other groups at 10 d of culture (P <0.05). The area of covered by the colonies were not significantly different between different groups (P>0.05). Caprine SSC colonies were found to have AKP activity. The number of colonies developed in three-step enzymatic digestion were significantly higher (P<0.05) a two-step enzyme and cold trypsin digestion, while the number of colonies developed in two-step enzyme clones significantly (P<0.05) higher than the cold trypsin digestion. The total cells and Thy-1 positive cells after antibodies plate were was significantly lower than other purification methods (P<0.05), while the total number of cells and Thy-1 positive cells after BSA+ laminin joint purified culture plate were the highest. The number of caprine SSC colonies after BSA+ laminin joint purified culture plate were the highest at 10 d of culture; however, area covered by caprine SSC colonies were not significantly different between different purification method (P>0.05). Caprine SSC colonies were found to have AKP activity. (2) The SSCs colonies were no major morphological differences and have AKP activity, and express VASA, integrins, Oct-4, and PLZF marker between three feeder layers at 4,7,12 d of culture. The area of colonies was the highest in GSC feeder layers group, while the number of colonies was the most in GEF feeder layers group. The number and area of colonies were both the smallest in MEF feeder layers group. Concentrations of key cytokines including GDNF, bFGF and IGF1 for improving SSCs growth were the highest, however, the concentration of LIF was lower compare with GEF and MEF feeder layers groups in GSC feeder layers group regardless of the culture time; no significant difference was observed between for concentrations of key cytokines between GEF and MEF feeder layers groups (P>0.05). (3) SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphological differences between the groups. The number and area of colonies were both the highest in the 40 μg/mL Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia and testis somatic cells was observed. Cultured germ cell clumps were found to have AKP activity regardless of the Vc dose. The number of Thy-1-and Oct-4-positive cells was the most in the 40 μg/mL Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 μg/mL was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the anti-apoptotic gene Bcl-2 and decreasing the expression of the pro-apoptotic genes Bax and P53. (4) The graft block weight treated and untreated HGF in renal capsule and the dorsal subcutaneous site were not significant (P>0.05) at 20 days after transplantation. The graft block weight treated HGF in renal capsule site was higher compare to untreated HGF in renal capsule site and in the dorsal subcutaneous site at 40 and 60 days after transplantation (P<0.05), no statistical difference was observed between other groups (P>0.05). RT-PCR and qRT-PCR showed that expression of undifferentiated and differentiated spermatogonia (Vasa, Oct4, Thyl and PGP9.5) have no significant change； however, expression of meiotic cells markers (STRA8 and SCP3) and post-meiotic cells (Prm-1 and Acrosin) markers were the strongest in HGF treatment group after transplantation into the renal capsule. (5) The single cell suspension using a three-step enzymatic treatment contained sertoli cells, germ cells, muscle-like cells and Leydig cells. Efficiency of SSCs differentiation in adding 10 ng/mL SCF+10-5 mol/L RA+50 IU/L FSH+1μmol/L T+basic medium was the highest by the cell ploidy analysis in 3D-SACS at 30,50 and 70 days after culture; and expression of HSC70t was also the highest in adding 10 ng/mL SCF+10-5 mol/L RA+50 IU/L FSH+ 1μmol/L T+basic medium; RT-PCR and qRT-PCR analysis showed that expression of undifferentiated spermatogonia marker had no statistical difference at 30,50 and 70 days after culture between four different groups (P>0.05); however, expression of PGP9.5 significantly greater in control group compared with the other groups (P<0.05) at 30 days after culture, no statistical difference was observed between other groups (P>0.05); no statistical difference was also observed between four different groups (P>0.05) at 50 and 70 days after culture; expression of c-Kit significantly lower in control group compared with the other groups (P<0.05) at 30 days after culture, however, expression of c-Kit in adding 10 ng/mL SCF+10-5 mol/L RA+50 IU/L FSH+1μmol/L T+ basic medium was the highest at 50 and 70 days after culture, and was significantly higher compared with the control (P< 0.05), but no statistical difference was observed between other groups (P>0.05). Expression of meiotic cells markers (STRA8 and SCP3) in the control significantly lower compared with the other groups at 30,50 and 70 days after culture (P<0.05). Expression of post-meiotic cells (Prm-1 and Acrosin) markers in the control were also significantly lower compared with the other groups at 30,50 and 70 days after culture (P<0.05). Conclusion:(1) 1-month goat was used as appropriate SSCs donor; three-step enzymatic digestion was the most appropriate; BSA+ laminin joint purified culture plate was considered to be the most appropriate purification method. (2) Goat GSC and GEF feeder layers were more appropriate be used as the feeder layers for SSCs culture in vitro. (3) The addition of 40 μg/mL Vc can maintain a certain physiological level of ROS, trigger the expression of the anti-apoptosis gene Bcl-2, suppress the pro-apoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro. (4) The spermatogenesis of graft block in the renal capsule site was better than in the dorsal subcutaneous site; and HGF can promote the formation of blood vessels, cell migration and immune protection of graft block in the dorsal subcutaneous and the renal capsule site after transplantation. (5) Efficiency of SSCs differentiation in adding 10 ng/mL SCF+10-5 mol/L RA +50 IU/L FSH+1μmol/L T+ basic medium was the highest by the several detection methods analysis in 3D-SACS at 30,50 and 70 days after culture. |
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