Testicular Infertility Treatment By Autologous Bone Marrow Mesenchymal Stem Cells Via Intra-testical Transplantation In Rabbits

Part Ⅰ Preparation ofinfertile animal model by local electron beamirradiation on rabbit scrotumOjective: To explore the feasibility of local single dose electron beamirradiating the scrotum of young adult male New Zealand white rabbits toprepare infertile animal model, which will be applicated to study infertlitytreament through intratestical transplantation by autologous bone marrow stemcell.Methods: Thirty of healthy5-mon-old male New Zealand white rabbitswere randomly divided into5groups. In experimental Group A, B, C and D,the rabbits testes were irradiated with single dose of6,8,10and12Gy,accordingly. The untreated rabbits served as the control group E, only layedon work station5min under anesthesia. After treatment for rabbits, thesurvival, vigour, eating, and properties of urine and shit were observed. At2rd,4th weekend postirradiation, routine tests of blood drawed through rabbit earsvene were applied to compared by quantity changes of white blood cells,hemoglobin and platelet; bone marrow harvested from unilateral tibia lumenunder anesthesia, was compared by the morphology of myeloproliferativedegree; at2rd,4th,6th,8th and12th weekend after treatment, one rabbit ineach group underwent bilateral orchiectomy after anesthesia. The testes wereroutinely sectioned and stained with hematoxylin and eosin, the sections werescored for the lack of meiotic cells and stage of spermatogenesis in at least400seminiferous tubule cross sections per testis. Then rate of hollowseminiferous tubules (RHSTs) was calculated and recorded as percentage ofshort of spermatogenesis. The RHSTs of experimental groups as well asuntreated control group were compared mutually with variable of time and dose, respectively.Results: All rabbits were survived. Blood urine, hematochezia, anddiarrhoea were taked place in none of the irradiated rabbits, whose vigour andeating showed the same as the normal control. There are no significantdiference in the count of white blood cells, hemoglobin and platelet in all thetreatment groups compared with the control group E at the2timepoints. Atthe2rd,4th weekend postirradiation, the bone marrow film of all treatmentgroups as well as the control group showed the similar active proliferation andthe proportion of granulocyte vs erythrocyte. Testes Histology and Evaluation:Seminiferous tubules contained meiotic cells, seminiferous epithelial cellarranged orderly, full spermatogenesis was seen in all tubules, and RHSTremained nil during the experimental period in normal controls. Theseminiferous epithelium showed varying degrees of damage in all treatmentgroups compared with unirradiated control. In general trend, RHSTs wereascending with the dose increasing in the same timepoint postirradiation, butSertoli cells and Leydig cells were survived normally. The seminiferousepithelium was restored gradually in group A and B with time. In the contrary,the damage of seminiferous tubules got more serious during6weeks andstable later in the group C and D which were showed with more significantdifference of RHSTs (p<0.05). The HRST of group12Gy was the highest withnear more than90%after6weeks postirradiation.Conclusions:1Local irradiation on rabbit scrotum with dose under12Gy did not createany serious whole body except the testical histology changes.2The seminiferous epithelium showed varying degrees of apoptosis andno influence on Sertoli cells and Leydig cells by irradiation under12Gy. Theseminiferous epithelium was restored gradully with time postirradiation inlower dose. In the contrary, the damage remains seriously with higher doseirradiation.3Local electron beam dose of12Gy, which once irradiated into scrotumof healthy5-mon-old male New Zealand white rabbits, is a optimal protocal for intratesticular transplantation with autologous BM-MSCs after6weekspostirradiation. Part Ⅱ The Study of transdifferentiation into spermatogonial stemcells(SSCs) induced by rabbit bone marrow mesenchymal stemcells(BM-MSCs).Objectives：In order to provide experimental fundament for autologousBM-MSCs transplantation in testes, we intended to explored the efficiencymethods of the isolation, culture, proliferation, induction, identification andlabeling for rabbits’ BM-MSCs in vitro; to observe the influence of localirradiation to the scrotum on the growth characteristics of BM-MSCs, todetermine if the rabbit’ post-induced BM-BMSCs have the capability oftransdifferentiation into spermatogonial stem cells(SSCs).Methods：New Zealand white rabbits of group D and group E in thepart Ⅰe xperimentwere applied. Following anesthetization and routinedisinfection procedures, bone marrow was taken from proximal tibia.Mononuclear cells BMSCs were isolated by density gradient centrifugationand purified by adherent separation to obtain a large number of BMSCs withuniform appearance.were were isolated, cultured, and proliferated in vitro byPercoll density gradient centrifugation combined with adherent separationmethod. MSCs were identified by dynamic observation of proliferationcharacteristics, HE staining, and immunohistochemical detection of cellmarkers(CD34, CD44). After incubated with SSCs inducing medium, P3MSCs were testified whether transdifferenciation gone for SSCs byimmunohistochemical detection of cell markers(CD117). RabbitMSCs-labeling positive ratio using BrdU for non-induced and induced MSCswere compared. Proliferation characristis and growth curves were obersevedand contrasted between non-induced and induced MSCs to exactly evaluatethe influence of irradiation. Finally, RNA expressions of Dazl, OCT-4，and c-kit relatived to germ stem cells were measured by RT-PCR to evaluted theinducation effects on MSCs.Results：The isolated stem cells grew adherently on the wall, with afibrous shape and strong proliferation ability. Primary cells showed colonygrowth, gradually confluence after10-12days culture, and speedily expansionafter passage. Growth curves and morphology of BM-derived stem cells inirradiation groups showed no difference with the normal control. Afterinduced medium culture, the generation of stem cells sizes increased, fusiformshape remained, along with thick lateral branch. some cells grew triangular orpolygonal. BMSCs grew typically difference colony and visible more celldivision phase. Immunohistochemically, noninduced cells showed positiveexpression of CD44and negitive expression of CD34to be MSCs, inducedcells appeared positive expression of CD29and CD117. RT-PCR showed anenhanced expression of the spermatogenesis-relative factor of Oct-4, Dazl andc-kit in induced MSCs. Immunohistochemistry showed no significantlydifference in labelling rates between induced and noninduce MSCsco-cultured with50μmol/L BrdU after72hours. Labelling rates both groupsreached more than80%.Conclusions：1Without no side reaction of irradiation on BM-MSCs growth, a singledose of12Gy to locally irradiate both testes is an efficacious protocol thatprovides a foundation to perform autologous BM-MSCs transplantation inrabbits.2The induced BM-MSCs maybe able to transfer to SSCs-like cells.3The induced BM-MSCs can be well lebelled by BrdU at50μmol/Lconcentration after72hours, that will be applied for tracing of MSCs in laterintratestical transplatation. Part Ⅲ Restoration of spermatogenesis by intratesticulartransplantation with autologous BM-MSCs in rabbitsObjectives：To evaluated efficiency of induced BM-MSCs to restorespermatogenesis in testical infertility by observing MSCs survival, migration,and testical histology changes as well as the expression of spermatogenesisraletived gene of Dazl, Oct-4, c-kit, and P53.Methods: The20healthy male New Zeland White rabbits were dividedinto4groups randomly(normal control, blank control, matched control, andMSC transplatation group), Normal control group only received shamirradiation; Under anesthesia, the othe rabbits of3groups were irradiated bothtestes locally with a single dose of12Gy performed by a electroni linearaccelerator to provide infertility animal models. Bone marrows were harvested2weeks later postirradiation, then MSCs were isolated, purified, and expanded.After induced5days in SSCs medium, passage3rd MSCs undergoneco-incabution with BrdU at50μmol/L concentration were to performautologous transplation via retes testis. After leblled with BrdU for72hours,MSCs of total0.2ml, at1×107cells concentration were transplate by a1mlsteril syring into bilateral testes of each rabbit in MSCs transplatation group,and equivlent volume of inducing culture medium were injected into rabbitstestes by the same way in matched ontrol group. Rabbits in blank controlgroup received no injection. After graft, there testes of each group wereobtained by orchiectomy at the timepoint2w,4w, and6w post-transplatation,respectively. The fixed testes sample were then to be embeded in paraffin,sectioned, and stained with hematoxylin and eosin. Restoration ofseminiferous epithelium were observed under light microscopy, and RHSTswere compared among the4groups. BM-MSCs survival and migration weretraced by BrdU immunohistochemical staining. Finally, RT-PCRsemi-quantitative analysis were performed to compare the expressions of Dazl,Oct-4, c-kit, and p53of fresh testes samples among all groups. All statisticalanalyses were performed using SPSS11.5.Data were analyzed using one-wayanalysis of variance (ANOVA)with least significant difference (LSD), and P-values less than0.05were considered statistically significant.Results: All rabbits survived during experimental stage. Transplantssmoothly, there was no infection occurred in local scrotum. Histology: Innormal control group, seminiferous tubules contained meiotic cells,seminiferous epithelial cell arranged orderly, full was seen in all tubules. Inblank control group, spermatogenesis arrested, seminiferous epithelial weredeleted, and tubules were near empty, but Sertoli cells existed normally. Inmatched control group, histological damage showed like blank control at earlystage, However, initiated restoration in few tubulus4to6weeks postgraft. InMSCs transplatatiom group, germ cells increased gruadully in seminiferoutubulus, cell layer is close to normal gradually from2weeks to6weekspostgraft, but sperm connot be seen until6weeks. Comparison of RHSTs: InMSCs transplatatiom group, RHSTs were reduced with time and significantlydifference at the timepoint2nd,4th, and6th weekend postgraft, reciprocally,(p<0.05). RHSTs of MSCs transplatatiom group reduced more significantlythan any othe group except that of normal group at the same timpoint. RHSTsdecreased down to20%after6weeks. BrdU tracing: MSCs exhibited aslightly messy arrangement and distribution in the center of seminiferoustubule cavity at2nd weekend postgraft, then migrating gradually into tubulusbasilar part from4th weekend to6th postgraft. RT-PCR showed an enhancedexpression Oct-4, Dazl and c-kit in MSCs transplant group than that in blank-and matched control group at6thweekend postgraft. Dazl：Relative quantitieswere similar in normal control and biank control, the other two groups showedsame quantities but higher than that of ahead two groups, that imply us mayberetinoic acid play a role on enhancing express of Dazl. Oct-4: It was similar ofOct-4expression in normal control and blank control, which showed higherthan of matched control and transplatation group, Oct-4expression was thelowest in matched group. The result suggested us perhaps retinoic aciddecreased the production of Oct-4. c-kit: There were similar of c-kitexpression in normal control and MSCs group, that exhibited much higherthan in blank and matched control, suggest us MSCs be able to converted to germ cells for sake of c-kit restoration. P53: Relative quantities of p53inMSC group was significantly lower then that of3groups, that reason maybeautolougous transplatation with MSCs inhibiting apopotisis and increasingproliferation of seminiferous epithelial.Conclusion：1Transfusion MSCs via rete testis is an easy, safty and efficient regimen.That needs higher concentration MSCs, stern steril operation, optimalpuncture angle and inject speed, etc.2The grafted MSCs via rete testis survived in siminiferous tubulus, thenmigrated into tubulus basilar part and settled to proliferate after2weeks sincetransplatation. RHST analysis showed significantly reduction, that suggestedautologous MSCs graft increasing restoration of damaged seminiferousepithelial.3Gene detection of related to primodial germ cells suggested that MSCsgraft in testis be able to convert to SSCs-like cells and inhibit apoptosis intubulus.4Spermatogenesis is a complex biological process, requiring a goodniche, various cytokines participation, and gene regulation, etc, which neededto be deeply researched in autologous BM-MSCs intratesticulartransplantation.