Study Of Goat Spermatogonial Stem Cells Culture In Vitro And Transplantation

Male germline stem cells—spermatogonial stem cells (SSCs)—self-renew and produce large numbers of differentiating germ cells that become spermatozoa throughout postnatal life and transmit genetic information to the next generation. And SSCs became new assisted reproductive technologies in the preservation of endangered wildlife and the management of commercial livestock. For the last decades, the achievement from the SSCs culture in vitro and transplantation development offered new ideas and technology support. This dissertation studied the isolation, purification, culture system optimization and transplantation techniques of goat spermatogonium in vitro respectively to construct an optimized long term culture system to facilitate in vitro manipulate studies for future. The results as following:1. The histosection of ram testes about 2-3 month old were made and observed, the morphology of germ cells were uniform in seminiferous tubules and did not enter the spermatogenesis. We have found a lot of PGP9.5 positive spermatogonium on basal membrane and it could be used culture in vitro. We have developed an ideal isolation method, two step enzymatic digestions. The first 0.1%collagenaseⅣ+0.1% hyaluronidase for 20 min and than 0.25% trypsin+0.04% EDTA+5μg/mL DNaseⅠfor 5 min,its enough to obtain germ cells and have optimal cell viability. 8.4±2.6×106 cells per gram testis were isolated and there was(19.7±5.3)% of PGP9.5+ spermatogonium and (23.8±3.6)% of c-kit+ spermatogonium.2. Three method, including Percoll density, centrifugal elutriation and plate method, for enrichment of PGP9.5+ cells were contrasted. The spermatogonium puirity (including the PGP9.5+ cells and the c-kit+ cells) to achieved above 82% by percoll density of 27-35% and panning method and PGP9.5+ cells were (63.6±3.2)% and (71.9±4.1)% respectively. The spermatogonium puirity by centrifugal elutriation were only 60%, but whit higher c-kit+ cells. The cells viability from panning method was the best.3. The study was about the development of SSCs in vitro. The sertoli cells and sprmatogonium(SCs-SSCs) co-culture system were established whitout growth factors addition and feeder layer. We found sermatogonium colonies formed in culture at d7 with addition of 2.5% FBS. The spermatogonium colonies in the SCs-SSCs co-culture system were at uneven distribution. The brid nest shaped colonies were abundant in perimeter of culture plat. We also found colonies to attach with the somatic cells at the center of culture plat. The cells in the colonies showed looser connect. and cells proliferation were slowly and the number gradually decreased after passages behind 3 passage. We have planted the cells from planning methods on the MEF feeder layer. The cells adherence was faster, cells proliferation began at 2-3d of culture and the cell clusters appeared at 5-6d culture. But cells proliferations were slowly without growth factors addition.4. the study have achieved a better culture system for long term maintenance spermatogonial stem cells using mouse stem cells for reference. In view of higher serum concentration speeded the proliferation of somatic cells; the 2.5% of FBS was used. The growth factors, GDNF, LIF and bFGF, were introduced into the culture system. The cells proliferations were promoted by growth factors and the shape and the size of colonies were also improved. GDNF could boost SSCs proliferation and increase coloies size. LIF was not significant improve SSCs proliferation, but for good shape colonies maintenance. The SSCs proliferation accelerated, colonies number increased by addition of GDNF and bFGF combination. It showed GDNF and bFGF synergistic action promoted the SSCs pool maintenance. The goat PGP9.5+ SSCs proliferated in vitro culture at least 3 monthe in MEF feeder layer containing 100ng/mL GDNF,10ng/mL LIF,10ng/mL bFGF and 2.5%FBS.5. Heterogenic transplantation was carried out using freshly isolated goat SSCs. The non-immuno suppressive mouse was used as recipient animal. Treated with 40mg/kg weight busulfan, the germ cells in mouse testis were destructed. After 4-6 week, it was used as recipient. Seminiferous tubules injection method was used and 50% of mouse testis was injected. Donor SSCs of goat stained with PKH26, it can obviously distinguish from mouse germ cells at mouse testis under fluorescent microscope. PKH26 can transfer to the daughter cells with cell division. SSCs of goat can adhesion to the basal membrane of mouse sreminiferous tubule and began proliferation. Donor cells in the mouse testis were decreased after 1 month after transplantation. Through histosection we found autologous germ cells of mouse began proliferate. Exogenous cells were rejected by the endogenous cells proliferation maybe.6. The goat testes with various size were collected from abattoir and to carry out injection test. Due to the difference in anatomy of ram and mouse testis, the methods on mouse injection is not suitable for ram testis. We have injected to the rate testis of ram testis. The injection sites were compared. The result show successful injection rate was the way of extra-testicular rete than intra-testicular rete and filling rate was same. Ultrasound guided injection and surgical injection was the same to the lager size of testes in successful injection rate, but surgical approch were better than in smaller size (8-25cm3) testis.