A Novel Method For Isolution, Cultivation And Identification The Female Mouse Theca-interstitial Cells And Granular Cells

Objective: To explore a facility, effective method of separating and culturingovarian theca–interstitial cells from granulosa cell in immatue mice ovary. Methods:The ovaries of21-25days-old female C57BL/6were collected. The ovaries werecleaned of surround marginal tissues by stereomicroscope and washed twice with icecold LSP medium. GCs were removal immediately by puncturing follicles from theisolated ovary with a25-gauge needle in the Falcon dish (Greiner, Frickenhausen,Germany). The residue tissue was reservated and washed twice with ice cold LSPmedium and finally incubated for90min at37℃in0.2ml/ovary ofcollagenase-DNase solution (4mg/ml of collagenaseⅣ,10mg/ml of DNase, and10mg/ml of BSA in M199medium).and then，the thecal-interstitial cells were transferredto culture plates containing in a known volume of McCoy’s5a medium, supplementedwith10%fetal bovine serum and10ng/ml androgen and cultured at37℃under awater-saturated atmosphere of5%CO2. The thecal-interstitial cells were detected byimmunocytochemistry, western blotting and hormone assay. At the same time, wealso throughed to add50ng/ml FSH or20ng/ml LH to detect the responsivity ofhormone secretion. Results: In the separated cells, the ratio of survival cells to thetotal cells was more than90%. Several cells could be visible as thin spindle orpolygons after24hours cell culture. Results: The result of immunohistochemistryshowed that the positive of AMH protein was lower than5%but P450scc protein washiger than95%；the hormone assay result showed that the level of testosteroneconcentration was649.22±12.3ng/dl(n=3) and the estrogen concentration was lowerthan10.7pg/ml(n=3). The concetration of testosterone in0.1U/ml group is significant higher than that of FSH groups and basic media group; The level oftestosterone concertration in0.5U/ml LH culture media was significantly higher thanthat of0.1U/ml LH culture media and basic media(p<0.05); there was on significanceamong0.5U/ml LH group,1U/ml LH group,2U/ml LH group; The level of estrogenconcertration was no detected in the all groups. Conclusions: The proceduredescribed by our study can be considered as a rapid, effective method to separate andculture immture mice ovarian theca-intertitial cells which barely mixed withcontaminated graunlosa cells.