| Objective A subcutaneous injection of Wistar rat's SSc(spmermatogonial stem cells)suspension and transplantation of neonatal Wistar rat testis fragments of different size were carried out.According to the growth and development of the grafts,to determine whether it would be a useful tool for investigations on the proliferative and developmental pattern of spermatogenic cells and the regulation mechanism of spermatogenesis.Methods1.The preparative method of Wister rat's SSc:The testis were aseptically isolated from neonatal Wister rat(7～9 day old)and placed into fiat plate containing 20 ml DMEM/F12 mediums immediately.After removal of the outer connective tissue layer and epididymides,the content of the tunica albuginea was squeezed with pincers,minced and transferred to a new vial containing 5 ml DMEM/F12.The pieces of tissue were mixed by repetitive pipetting with a 10 ml pipette.The sample was sedimented for 3-4 min at room temperature,the supernatant were discarded and the purify contorted seminiferous tubules were yieled.Cellular dissociation was accomplished by two-step sequential enzymatic digestions.In brief,the seminiferous tubules were first digested with type I digestive juice in a shaking water bath 100 per min at 33℃for 15 min.Isolation of seminiferous tubular fragments from interstitial cells was achieved by repeated sedimentation at unit gravity.In a second digestion step,tubule fragments were incubated with typeⅡdigestive juice under the same condition for 30 min.If necessary,the procedure may repeat one time until a single-cell suspension was achieved.Then add the DMEM/F12 medium supplemented with 10%fetal bovine serum into the vial to end digistion.The cells were washed twice in the DMEM/F12 and filtered in the 200-eyes and 300-eyes cell filtration resprectively.The SSc suspension originated from the testis was achieved.According to the different adhesiveness,5 ml(1 ml per grade)Percoll uncontinuous density gradient were put into 10 ml centrifuge tube by turns to purify the SSc.The SSc suspension that was located in the upper centrifuge tube containing 5 ml Percoll uncontinuous density gradient were centrifuged for 20 min at 1400 cycle per minute.There are 4 belts lacotaed in close togethe gradient in Percoll.Take the cells out from the the third belt carefully and shift it in the 5 ml centrifuge tube supplemented with 2 ml PBS.The celle were centrifuged again for 3 min at 1000cycle per minute.Then discard the supernatant and add 2 ml DMEM/F12 mediums into the centrifuge tube.A trypan blue solution was injected into 1 ml SSc solution.Total cell numbers,live cell and dead cell were assessed using a bright-line hematocytometer respectively within 3 minute.2.Homotransplant:One testis obtained from neonatal Wister rat(7～9 day old)was divided into two halves in DMEM/F12 containing 1000u/mL benzylpenicillin and 1g/l Streptomycin.Four pieces of testis tissues and 1ml SSc solution(the number of cell 4×10~6个/ml,survival rate＞95%)immediately were grafted into the back skin of castrated Wister rat(8～12 week old) respectively.A total of 8 Wister rats,clear grade,weight 180～220g,were divided into two groups randomly:group A(n=5),which were operated with SSc solution; group B(n=3),which were operated with testis tissue.The growth and development of grafts were observed after experimentation.At postgraf 8 weeks,the grafts were token from the back skin of the recipient and fixed in Bouin fixative at 4℃for 24h followed by dehydration in 70%ethanol.The grafts were washed in xylenes,blocked in paraffin and sectioned at 8 um.After sectioning,slides were deparaffinized,rehydrated,and stained with hematoxylin and eosin.The grafts were evaluated under light microscopy.Result1.The preparative method of Wister rat's SSc:A total of 6 experiments in which we count the total cell number and the dead cell number were perfomed.The total number of cell,the number of live cell and the number of dead cell was(6.68±1.36)×10~6,(6.06±1.29)×10~6,(0.61±0.10)×10~6 in one testis respectively.The average percentages of live cell were(90.73±1.39)%. There are 4 belts lacotaed in close togethe gradient in Percoll after the cells solution were centrifuged in the Percoll uncontinuous density gradient contrifugation and isolation.The first belt,11%-19%Percoll gradient,the major cell is intercellular substance,cell debris and slight dead cell;The second belt,between 19%-27%Percoll gradient,the major cell is Sertol cell; The third belt,between 27%-35%Percoll gradient,the major cell is spermotogonia and the number of cell is very large;The forth belt,between 35 %-43%Percoll gradient,the number of cell is very small.The spermotogonia was identified by the characterized shape and size under light-microscope.2.Homotransplant:The transplantation position did not detecte red swelling of the skin and suppuration.The grafts commence to grow after 6 w but vanish after 11 w in group B.In group A,the grafts growth was not observed from the beginning to the end.The grafts histological analysis demonstrated the structure of seminiferous tubules,spermatogonial stem cell,meiotic germ cells and a lot of lymphocyte infilitrating in testis interstitral under a light microscope. elongating spermatids was dectected in some seminiferous tubules and some seminiferous tubules degenerated.Conclusions:1.Wistar rat SSc can be isolated and purified effectly by the methods of sequential two-step enzymes digestion and Percoll uncontinuous density gradient centrifugation2 The reason that Wistar rat SSc grafted into Wistar rat can not survive and proliferate may be due to the microenvironment defiency.3 The neonatal Wistar rat testis tissue can continuously grow and develop under the back skin of Wistar rat.The spermatogonial stem cells of testis tissue grafts can finish a complete spermatogenesis and develope into spermatid.But,it is necessary for the funcation of the spermatid to be further assessed.
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