Purification And Long-Term In Vitro Cultire Of The Bovine Spermatoginial Stem Cells

Spermatogonial stem cell (SSCs) is the unique adult stem cells which can deliver the genetic information of parent to the descendant by natural fertilization. Through the self-renewal proliferation and differentiation, it can maintain the whole spermatogenesis balance and progressing. The initial study about SSCs began in the mouse, and as yet there has been successfully developed the methods of isolation and purification, long-term in vitro culture, SSCs transplantation and in vitro differentiation of the mouse SSCs, which has not only established the solid basis for the study about mouse spermatogenesis, but also lightened the future of the research on spermatogenesis of other mammal. However the study on the SSCs of domestic animal is lagging behind relatively, although it has been made some important advancement. According to the published reports on isolation and purification, long-term in vitro culture, SSCs transplantation and in vitro differentiation of the mammalian SSCs, also on the basis of the in vitro culture systems of rat, sheep, goat and pig SSCs establish in our lab, this preliminary study focused on isolation and -purification, long-term in vitro culture, in vitro differentiation and transplantation of the SSCs of black-white diary cattle.1. Isolation and purification of Spermatogonial stem cells of domestic black-white diary cattleThe 6-8 months black-white diary cattle were used in this study. The method of three-step enzymatic digestion was used to prepare single cell suspension.3×107 (n=5,±.5 × 107) cells were obtained from 3 g tissues of testis. Following differential plating on gelatin, collagen and Laminin coated plates respectively, highly purified bovine spermatogonial stem cells (bSSCs) were obtained. To determine the enrichment efficiency of each step, the smears of single cell suspension (SCS), collagen I non-binding cells (CNB) and Laminin binding cells (LB) were prepared. After immunofluorescent staining, the total cells and the bSSCs which was labeled with PLZF antibody were counted. It was found that the bSSCs ratio in SCS was 24%±5%, in CNB was 50%±3%and in LB was 83%±7%(n=5) respectively. This result was met the purity of in vitro culture condition, and provided the basis for long-term culture of bSSCs.2. Long-term culture and preservation of spermatogonial stem cells of black-white diary cattleAccording to the long-term in vitro cultural method of rodent SSCs, a long-term culture system of bSSCs has been established. The culture system can support the bSSCs in vitro expansion for over 31 passages with maintaining its morphological and genetic characteristics. The cell number counting of 3 continuous passages indicated that the bSSCs doubling time was about 5-7 days in this culture system. To determine the optimal GDNF concentration for this culture system, this study measured the effect of GDNF in different concentration (20 ng/ml,30 ng/ml,40 ng/ml) by counting the growth number of bSSCs after 7 days in vitro culture. The statistical results illustrated that the bSSCs number was growing with enhancement of GDNF concentration. The effect on increasing cell number was significantly (P<0.01) while using 30 ng/ml GDNF compared with 20 ng/ml group. However the increase was not significant (P>0.05) compared between the groups of 30 ng/ml and 40ng/ml. By considering the elements of cost and operation, we selected the 30 ng/ml GDNF as the optimal concentration for in vitro culture of bSSCs in this system. In summery, the method for long-term in vitro culture of bSSCs has been established by using the serum free culture system with growth factors and feeder layer. The achievement lay a foundation for further study of bSSCs.3. Identification of long-term cultured spermatogonial stem cells of black-white diary cattle via marker moleculesFor identification of marker molecules of bovine spermatogonial stem cells, immunofluorescent staining on the frozen section of bovine testis was carried out for verification if CDH1 is a marker molecule of bSSCs. The PLZF has been reported widely as the marker molecule applied on identification of spermatogonial stem cells of many mammalian animals included bovine. After labeling bSSCs with PLZF on the frozen section of bovine testis, immunofluorescent staining was performed by using CDHl antibody. The results showed that CDH1 distribution was overlap with PLZF in the As, Apr and Aal spermatogonia near the basement membrane. The result confirm that CDH1 could be used as a molecular marker for identification of bSSCs. RT-PCR analysis reveal that Thy1 and CDH1 were transcribed in bSSCs. The characterization of long-term in vitro cultured bSSCs was accomplished by checking the marker molecules of bSSCs by immunofluorescence double-label staining and it was found that PGP9.5, Gfrα1, Oct4, Thy1 and CDH1 were co-expreesed with PLZF respectively in the long-term cultured bSSCs. These results illustrated the conservative expression of these marker molecules in bSSCs, and confirm that the long-term cultured bSSCs maintained the character for expression of the marker molecules. It is also provided the reference for further study of bSSCs.4. The functional analysis of spermatogonial stem cells of black-white diary cattleFor verifying the differentiation ability, the long-term cultured bSSCs were induced for differentiation. This study performed the in vitro inducing differentiation and SSCs translating experiment. According to the report on inducing differentiation in vitro by Dym group, and the method of inducing differentiation of sheep SSCs that established by our lab, the medium containing the FBS and SCF was used to induce the in vitro cultured bSSCs for 10 days. After the induction, SCP3 positive spermatocytes were detected in the differentiating system by immunofluorescent staining. Furthermore, the cryopreserved bSSCs passaged to 15 generations were transplanted into the testes of cattle-yak, which is sterile and used as recipient of bSSCs transplantation. After transplantation for 1 year, the frozen sections were prepared from the testis, and were analyzed by histochemical staining. Microscope observation revealed that the seminiferous tubules of the transplanted testis were filed with cells, whereas the seminiferous tubules of un-transplaned testis were empty. Real-time PCR analysis of the transplanted bSSCs expressed PGP9.5, SCP3 and Tnp2 genes in the cattle-yak testis. These results illustrated that the long-term cultured bSSCs maintain physiological function. It is the first time to approve that the sterile cattle-yak can be the natural recipient candidate for bSSCs transplantation.