Effects Of INSL3 On Cultural Mouse Gubernacular Testis Cells

Objective:Many studies indicate that high incidence of male sexual differentiation and devolopment of testicular tissue is related to the devolpment of testis and testicular descent. The gubernaculums play an essential role in the complex mechanism of devolpment of testis and testicular descent. The INSL3 gene expresses widely in human, and the polypeptide belongs to insulin-like family, which is essential for the development of gubernaculums during the first of the two-stage process of testicular descent. So far, the mechanism of the role of INSL3 on gubernaculums is controversial. The cultured mouse gubernacular cells were treated with INSL3 in our experiment. To investigate the effects of different doses of insulin-like factor 3(INSL3) on the proliferation and contraction of mouse gubernacula cells in vitro.Methods:Gubernacula testis from 3-day-old mice were removed by operating magnifier and placed into phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with an enzyme solution containing typeâ… collagenase (1mg/ml). The cultures were grown in 5% CO₂ and 95% air in phenol red-free DMEM containing 5% charcoal dextran-treated FBS. After 6⁸ days, primary cells were transferred into each wall of a 6-well culture plate. Cell viability was measured by trypan blue. Cell morphology was observed by HE stain. Subcultured cells were devided into five groups randomly, that is, experimental groups 1⁴: given 0.02ng/ml, 0.20ng/ml, 2.00ng/ml, 20.00ng/ml INSL3 respectively and normal control group. After be treated different hours, the gubernacular cells were harvested to observe the morphology, proliferating and expression of F-actin by CCK-8, immunofluorescent technique and FCM(Flow CytoMeter).Results:1. Most of the primary cells were fibroblasts, and only a few epithelioid cells. Fibroblasts were closely, radial, knitted or circinate. The cell bodies were spindle-shaped, irregular or triangle. After transfer of culture, gubernacular cells were mainly composed of fibroblasts and highly homogeneous . The gubernaculum cell viability was about 90%.2. Gubernaculum cells of normal group showed persistent proliferation, especialy after 24h. Similar to normal group, the experiment groups also displayed persistent proliferation. The rates of proliferation were higher in INSL3 groups than that in normal groups (P<0.05).3. In the normal group most fluorescein-labelled substance was scattered in the cells, weak fluorescence at cellularmembrane, and few F-actin filaments were observed with irregular alignment. Compared with the control group, the filaments of F-actin in the experimental groups were thicker, longer and showed in longitudinal arrangement, and the intensity of fluorescence was significantly increased.4. The expression of F-actin were obvious dosage-effect and time-effect correlations(P<0.05). In low dose groups (0.02ng/ml, 0.20ng/ml) of 24h groups, the expression of F-actin decreased slightly, but after the dose upto 20ng/ml, the expression of F-actin increased obviously. In high dose goup of 48h groups, the expression of F-actin increased obviously. Compared to 24h groups, the F-actin expressiones were upregulated in all dose groups of 48h groups.Conclusion:1. Most of the cultured gubernaculum cells were fibroblasts and highly homogeneous. The cell viability was high.2. INSL3 can stimulate the proliferation of gubernaculum cells with dosage-effect and time-effect correlations.3. INSL3 can stimulate reconstitution of cytoskeleton with dosage-effect and time-effect correlations and aslo cause descent of testis gubernaculums in mice.