Study On Isolation And Vitro Culture Of Yoshitomi Tilapia Spermatogonial Stem Cells

Spermatogenesis is a complex and subtile process.Spermatogonial stem cells（SSCs）coordinated by neuroendocrine and environmental factors well arranged undertakeself-renewal and differentiation for the normal spermatogenic in the male gonads. Spermas to give the carrier of genetic information to offspring is the best material for geneticmaterial.The germline stem cells is a prerequisite to from sperm cells.Genetic engineeringtechniques can be used to produce feed conversion rate,disease resistance,and has a highquality protein transgenic animals.People know something about the general process andregulating mechanism of spermatogenesis,but it is still unclear for most animals.So in vitroculture SSCs with simulate male gonads microenvironment play an significance role inreveal the process and regulating mechanism of spermatogenesis. For the vitro productionof sperm and the sperm as a carrier of transgenic cells,tissue and animal provide materialsand technology. Not only can protect the genetic data of endangered species also hasprovide technical basis for new transgenic animals with commercial value. Great progresshas been made in research of mammalian SSCs isolation and cloning in vitro,but fish SSCsis limited to zebrafish and medaka,ect.So far, domestic tilapia cultured SSCs vitro studiesrarely reported.Tilapia SSCs isolation, cloning and cryopreservation methods still need tofurther optimized and discussion. To investigate the fish SSCs cultured in vitro, maleYoshitomi tilapia as model animal, spermary as experiment materials do the followingresearch.1, Obtaining SSCs and SCs with different kinds of digestuve fluid and differentseparation method, purifieed by differential velocity adherent method,cloning method andmechanical method,the CO₂and Hepes concentration were modified to study the effect ofTilapia SSCs and SCs cultured,and then morphology observation, HE,oil red O and AKPwere used to preliminary identification the SSCs and SCs;2, Tilapia SSCs were seeded inthree kinds feeder layers and to select a feeder layer for proliferation of TilapiaSSCs.tilapia spermary extract,zebrafish embryo extract,EGF or bFGF were respectivelyadded in the SCs feeder layer to study the effect of SSCs proliferation,the growth curvewere detected to set up the amplification system of tilapia SSCs;3Cryopreservation oftilapia SSCs and SCs with different frozen stock solutions and cooling methods,and thenrecovery the cell in the same method,the recovery rate was detected. Finally,choice to freezing medium and cooling way for cryopreservation of tilapia SSCs and SCs.The resultsshowed as follow:1.With trypsin digestion Yoshitomi tilapia testis,the resulting total number of cell andliving cells were significantly higher than the groups of0.1%collagenase â…£and0.04%EDTA·Na₂（P<0.05）,and the first adherent time was significantly shorter compared with0.1%collagenase â…£and0.04%EDTA·Na₂（P<0.05）. There were no difference in totalnumber of cell,living cells and first adherent time of the trypsin group and the group ofcombination of enzymes（collagenase and trypsin,similarly hereinafter）（P>0.05）,but thedigestion time was shorter than others groups.So with trypsin could efficient and rapidseparation to Yoshitomi tilapia testis cells.Using L-15+0.1mmol/L β-mercaptoethanol+2mmol/L glutamine+1mmol/L non-essential amino acids+1%tilapia serum+10%FBSculture of isolated cells. As more hybrid cell in the tissue culture,further purified SSCs andSCs was difficult,and therefore should not be used. Added5%CO₂was not conductive togrowth of Yoshitomi tilapia SSCs and SCs.The previous3d appropriate growth in culturemedium of pH7.2-7.4,but with the extension of culture time,higher pH more contribute toproliferation of SSCs and SCs.The mainly cell were SSCs and SCs with trypsin digestion Yoshitomi tilapia testisafter identified, The morphology observation of microscope and HE staining showed thatSSCs were round or oval,large volume,abundant cytoplasm,and dependent on SCs withcolony form.Oil red O staining indicated that there was almost no lipid droplet in the SSCscytoplasm, showed AKP positive.Whereas SCs were polygon or triangle spreaded in theplate bottom, a large numuber of lipid droplet contains in the SCs dyed jacinth,and AKPnegative with no staining.2. Respectively purity was85%and88%of SSCs and SCs had been obtained bymechanical method.There was60%-70%stick wall of SCs to subcultured,generally3-4dfor generation,SCs pass five of total;The need to subcultured when had a large numbercolony of SSCs in the SCs feeder layer,primary culture5-6d was the best time of SSCssubcultured. Generally3-4d to generation because of slow proliferation of SSCs,has spreadto the fifth generation,replaced half culture medium for every other3d.The SSCs and SCskept to the original form in the process of subcultured. SCs feeder layer was significantlypromote proliferation of SSCs than mouse fetal fibroblast cell and rat pancreatic epithelialcell（P<0.05）,the latter two feeder layer were almost no effect for proliferation of SSCs,butwith the culture time extend to inhibit their growth.Using basic culture medium L-15+1%tilapia serum+10%FBS culture,SCs as feeder layer,and added different concentrations of tilapia spermary extract, zebrafish embryoextract,EGF or bFGF to amplification of Yoshitomi tilapia SSCs. The absorbance of cellswere determined by CCK-8assay in the1d to7d. Further, the growth curves of cells weredrawn. The results showed that all of the additives could increase the proliferation ofYoshitomi tilapia SSCs. And supplement of10%zebrafish embryo extract,10ng/mL EGFand10ng/mL bFGF could significantly increase the proliferation of Yoshitomi tilapia SSCs.10ng/mL EGF and10ng/mL bFGF promote proliferation effect was most obvious,and10ng/mL bFGF slightly better than10ng/mL EGF.3.The SSCs of Yoshitomi tilapia had a well cryopreservation result withFBS:DMSO=9:1of cryopreservation media and cooling condition which was4℃（30min）â†'-20℃（1h）â†'80℃（12h）â†'put into the liquid nitrogen, the average recovery rate was78%. With the cooling condition above, FBS:DMSO=9:1and FBS:DMSO:culturemedium=1:1:8of cryopreservation media could be used to store the SCs of tilapia, theaverage recovery rate was82.5%and79%, respectively.