Enrichment, Transplantation And Culture Of Mouse Spermatogonial Stem Cells And Mechanisms Underlying The Effect Of GDNF On Spermatogenesis

Spermatogonial stem cells (SSCs),the only stem cells that are capable of transmittinggenetic information to subsequent generations in adult animals,have abilities to self-renewand differentiate into spermatozoa. Therefore, SSCs are not only the study object of stemcell biology, but also the valuable resource of in vitro spermatogenesis, gene analysis andfunctional genomics. Brister et al (1994) established the SSC transplantation technique,and Nagano et al (2001) and Hamra et al (2002) successfully in vitro manipulated SSCs byusing different vectors and produced transgenic mice and rat after transplantation back intorecipient testes respectively. These studies provided a new method for delineatingspermatogenesis and making transgenic animals. However, the long-term culture of SSCshad not been achieved in a long period of time. Exhilaratingly, it has been recently reportedthat SSCs derived from mouse and rat could be maintained in vitro for more than sixmonths and sustain stable proliferation. In present study, we first established a system ofisolating, enriching mouse SSCs, and successfully transplanted the enriched SSCs intorecipient males and obtained donor-derived offspring, and then cultured the selected SSCsfor about one month in our serum-free defined medium with MEF feeder cells.Three mouse strains ICR, DBA/2 and KM, and one transgenic mouse line ICR/ EGFPthat expresses EGFP gene ubiquitously in all cell types of the body, were used in this study.Testes were isolated from 4-to 5 days postpartum (dpp;day of birth is designated as 0 dpp)male pups, and testis cells were collected by two-step enzymatic digestion according toprovirously described with some modifications. Digestion solution I consists of 2 mg/mlcollagenase type IV/V, and 200 μg/ml DNAse in Ca2+-and Mg2+-free PBS. Digestionsolution II contains 2 mg/ml collagenase type IV or V, 200 μg/ml DNAse and 2 mg/mlhyaluronidase in serum-free DMEM (Dulbecco's Modified Eagle Medium). Freshlyisolated testes were first washed 3 times in PBS. After decapsulated, testis samples weretreated with 10 volumes of Digestion solution I at room temperature for 3-to 5 minutesaccompanied by gentle agitation until the tubules were separated, and then washed 2 timesin 10 volumes of PBS by centrifugation at 170g for 3 minutes. For the second digestion,collected specimens were treated with 5 volumes of Digestion solution II at roomtemperature for 2-to 5 minutes with vigorous aspiration until tubular clumps wereinvisible. The dissociated cell suspension was added 5 ml of PBS or DMEM medium orDMEM/F12 medium containing 10% FBS, filtered through a nylon mesh with 60 μm poresize. The filtrate was washed 2 times by centrifugation at 200g for 5 minutes each time,and the pellet was finally resuspended with adherence selection medium, plated on0.2%(w/v) gelatin-coated tissue culture flasks (25cm2) at 2х105cells/cm2, and cultured at37°C in an atmosphere of 5% carbon dioxide in air.Testis somatic cells including Sertoli cells and Leydig cells were reported to bedetrimental to SSC self-renewal. To avoid the negative effect, it is crucial to removesomatic cells as soon as possible and use enriched SSCs for in vitro culture. Thus, we usedthe adherence selection medium containing 10% FBS, 2 mM L-glutamine, 100 unit/mlpenicillin and 100 μg/ml streptomycin in DMEM/F12 to enrich SSC population throughdifferential adherence selection, and observed that Sertoli cells attach to the bottom offlasks pre-coated with 0.2% gelatin more rapid than germ cells, which just loosely attach tothe somatic cell layer and are easily resuspended following vigorous pipetting. Afteragitation, therefore, floating cells were recovered and transferred to fresh flask alsopre-coated with 0.2% gelatin. The adherence selection was conducted 3 times in succession,and time of transferring floating cells to next flask is 1st, 4th and 20th hour after initialplating respectively. Whether the third selected cells need to transfer is dependent on theremoval status and proliferation state of testis somatic cells.To validate the efficiency of differential adherence selection, we used flow cytometricanalysis (FCA) to analyze the number and percentage of SSCs in third selected cells incomparison with the result of FCA for freshly isolated testis cells. Floating cells collectedby vigorous agitation were centrifuged, trypsinized, filtered through a nylon mesh with 60μm pore size, and then stained with antibodies against Thy-1, EP-CAM, GFRα1 and soforth, which have been shown to be SSC markers. FCA results indicated that thedifferential adhesion selection could increase the SSC population by 20.3, 11 and 14.8folds when the three antigens were analyzed in FCA assay respectively. Thy-1, EP-CAMand GFRα1 have been previously used as markers to sort SSCs from testis cells inmagnetic activated cell sorting (MACS) and fluorescent activated cell sorting (FACS). Ourresults suggested that the differential adhesion selection is very effective in comparisonwith MACS and FACS, and that the enriched SSCs population is enough to establish thecultures of SSCs after another adhesion selection or directly on MEF feeders because theSSC number is infrequent in the testis, and no specific SSC markers have yet identified.To ascertain the selected cells contain SSCs and maintain their stem cell activity, weperformed transplantation assay. ICR male mice were used as recipients, and treated withbusulfan (40 mg/kg b.w) at 4– to 5 weeks of age to deplete endogenous germ cells in thetestes. Donor cells were isolated and enriched from EGFP transgenic ICR pup males. Atthe time of transplantation 4– to 6 weeks after busulfan treatment, the recipient mice wereanesthetized intraperitoneally by Avertin injection (640 mg/kg b.w.), and approximately 15μl of selected cell suspension prepared with adherence selection medium was transplantedinto the seminiferous tubules of one recipient testis through the rete testis, leaving theanother as control. Donor cell concentration was adjusted to (0.5 – 100) × 103/μl. Twomonths after transplantation, recipient testes were recovered, and SSC-derivedspermatogenic colonies were visualized by observing the fluorescence under UV light. Toexamine whether germ cells generated in recipient testes were functionally normal, 5recipients were mated with wild-type ICR females 3 months after transplantation, and tworecipients gave birth to 17 offspring, among them 3 EGFP heterozygous pups (one maleand two females) were gained at 113 days after transplantation. The donor origin of pupswas confirmed by fluorescence under UV light. Taken together, these results indicated thatour selected cells contained SSCs and maintained SSC activity.Previous studies have been demonstrated that serum has a detrimental effect on theSSC maintenance by inducing differentiation. Therefore, we designed and prepared aserum-free defined medium for SSC culture after tried again and again. The SSC culturemedium consisted of Stem Pro-34 SFM supplemented with Stem Pro nutrient supplement,25 μg/ml insulin, 100 μg/ml transferring, 60 μM putrescine, 30 nM sodium selenite, 6mg/ml D-(+)-glucose, 30 μg/ml pyruvic acid, 50 μM 2-mercaptoethanol, 100 nM ascorbicacid, 10 μg/ml d-biotin, 1 μl/ml DL-lactic acid, 5 mg/ml bovine albumin, 2 mML-glutamine, minimal essential medium (MEM) vitamin solution, MEM nonessentialamino acid solution, 10 ng/ml recombinant human bFGF, 20 ng/ml recombinant rat GDNF,200 ng/ml recombinant rat GDNFsRα1(GFRα1)/Fc chimera. The selected cells, namelyenriched SSC population, were directly cultured on MEF feeders inactivated with 10μg/mlmitomycin C or after another adhesion selection with above medium, and maintained at37°C in an atmosphere of 5% carbon dioxide in air.In order to further eliminate somatic cells, instead of regular passage which usestrypsinization, only the floating germ cells were transferred to new plates with MEFfeeders by using pipetting method. After another 2-to 3 passages with MEF feeder cells,SSCs started to steadily expand along with the disappearance of somatic cells. Generally,the SSC colonies could be observed 3-to 4 days after transferred to MEF feeders. Thecolonies were usually about 4-to 80-cell size, looking like aligned type of undifferentiatedspermatogonia or a large cell mass with an irregular contour connected by cytoplasmicbridges. These phenomena were reproducible, and similar cultures have ever beenestablished from ICR, DBA/2 and KM origin. However, the establishment of SSC culturesfrom DBA/2 and KM strains is easier than that from ICR origin. We have successfullycultured SSCs for more than 20 days in 40 separate experiments, in which the longest timeis 28 days with 5 passages on MEF feeders. The in vitro culture of SSCs from KM originwas the first time in the field of SSC study.In order to testify the SSC identity of our cultured cells, we first detected some knownSSC markers including Oct4, GFRα-1, c-Ret and NCAM, by means ofimmunocytochemistry. The result of immunofluorescent staining showed that all cells incultured colonies were positive for these markers, indicating that cultured cells belong toSSCs.To confirm our hypothesis, we detected the expression of SSC marker genes againincluding oct4, sox2, GFRα1, ngn3(a marker of undifferentiated spermatogoina), c-Ret,Zfp145, piwi12, c-kit, Dazl, pum1, pum2. Piwil2 potentially play a role in SSCself-renewal;GFRα1 and c-Ret are the co-receptor and membrane receptor of GDNFrespectively;Zfp145 encodes the promyelocytic leukemia-associated protein (PLZF),which is a transcriptional repressor and probably one of the responsive factors for GDNFpathway. RT-PCR analysis showed that Dazl, a germ cell-specific gene, pum1 and pum2,two pre-meiotic germ cell markers, are all expressed at mediate levels, but c-kit is at alower level, which indicated that some of SSCs maybe automatically differentiate and losetheir phenotypic characteristics of SSCs. However, higher levels of expression of oct4,piwil2, GFRα1, c-Ret and Zfp145 as well as the low level of ngn3 expression decipheredagain that our cultured cell clumps were indeed SSCs. So, we have for the first timeestablished a long-term culture system of SSCs from KM mouse strain. Importantly, wededected the expression of oct4 and sox2 in cultured cells, suggesting that Oct4/Sox2regulatory complex possible play a crucial role in the maintenance of SSC self-renewal.Our study also indicated that GDNF is a central factor for the self-renewal of SSCs,and that GFRα1 and bFGF are also indispensable to SSC maintenance, implying thatGDNF and FGF signaling pathways play central roles in the SSC self-renewal. On thecontrary, LIF/gp130 signaling pathway is possible dispensable for the maintenance andproliferation of SSCs in vitro because the culture medium with or without LIF have noeffect on the formation and proliferation of SSC colonies.In conclusion, we have established a selection and culture system of mouse SSCs,including a method of differential adherence selection to enrich germ cells, a serum-freedefined medium, MEF feeders, transplantation assay and SSC marker analysis. Thissystem can be used as a paradigm to enrich and culture SSCs from other mammals.However, the mechanisms underlying SSC self-renewal and the optimization of long-termSSC culture conditions need to further investigate. SSCs are not only a valuable resourcefor making transgenic animals;their in vitro induced differentiation is also a important wayfor elucidating spermatogenesis. Especially, the derivation of SSCs from human will be ofgreat value for clinical applications in the future.In addition, we also conducted the study of GDNF effect on immature Sertoli cellproliferation as well as its possible pathway(s) by which GDNF signals. I noticed that thenumber of reports related this area was very limited, and the signaling pathways have notbeen characterized. Therefore, we used highly purified Sertoli cell primary culturesprepared from 4-to 5-day-old mouse testis to investigate the expression of GDNF, itseffect on Sertoli cell proliferation, and possible signaling pathways mediating the effect.GDNF expression in Sertoli cells was detected at both RNA and protein levels bysemi-quantitative RT-PCR and immunocytochemistry after treatment with FSH,testosterone and β-estradiol in different concentrations or within different time spans oftreatment. The results indicated that the expression of GDNF in Sertoli cells wasup-regulated by FSH, but both testosterone and β-estradiol have no effect on the expressionlevel of GDNF. Thus, we believed that the expression of GDNF in immature Sertoli cells isonly regulated by FSH.In order to confirm the effect of GDNF on Sertoli cells, we first evaluated the viabilityof cultured Sertoli cells by MTT supravital staining. Results indicated that GDNF at dosesof both 10 ng/ml and 20 ng/ml all promoted the viability of Sertoli cells significantly, and10 ng/ml GDNF in combination with 50 ng/ml FSH increased the absorbance unit to ahigher level. We then enumerated the proliferating Sertoli cells by BrdU labeling, andfound that the number of Sertoli cells in S-phase is dramatically increased in culturestreated with GDNF only and GDNF in combination with FSH. These results indicated thatGDNF promote the viability and proliferation of Sertoli cells in vitro, and there is asynergistic effect of these two factors on Sertoli cells.To elucidate the mechanism of GDNF controlling immature Sertoli cell proliferation,we detected the expression of GDNF receptor subunits in Sertoli cells by RT-PCR andimmunocytochemistry. Results indicated that immature mouse Sertoli cells express GFRα1and NCAM, but not Ret, implying GDNF signals through Ret-independent pathway. Inorder to validate this proposition, we performed antibody blocking experiments, andrevealed that the percentage of proliferating Sertoli cells in cultures with GDNF andanti-NCAM antibody was significantly reduced, suggestting that GDNF indeed signals viaGFRα1/NCAM pathway.We, therefore, concluded that GDNF is an autocrine growth factor produced in Sertolicells and regulated by FSH, and plays an important role in Sertoli cell proliferation viaGDNF/ GFRα1/NCAM pathway.