Influences of Temperature on SCF/c-kit Prolifertation Pathway and Cell Cycle of Rat Spermatogonial Cells Cultured in Vitro
|School||Zunyi Medical College,|
|Keywords||rat spermatogonial cells in vitro c-kit cell cycle gene mutation|
Objective:To investigate the influences of temperature on SCF/c-kit proliferation pathway and cell cycle of spermatogonia through rat spermatogenic cells cultured in vitro at different temperatures, to provide basic research data for the mechanism of spermatogenic impairment due to body cavity temperature (37℃).Methods:The isolated spermatogenic cells from adult male SD rat using mechanical methods and enzymatic digestion were cocultured with sertoli cells in incubator at 32℃and 37℃to observe the morphologic changes and growth characteristics every day. At 8th day cocultured in vitro, type A spermatogoni as were separated by gradient centrifugation and differential adhesion method. The cell cycle was detected by flow cytometry.Expressions of mRNA and protein level of c-kit、PI3K、cyclinD3 in SCF/c-kit proliferation pathway were detected by real time- polymerase chain reaction(RT-PCR) and western blot assay respectively. The exon sequences of 9、11、13 in c-kit gene were detected with sanger dideoxy.Results:At the 1st cocultured in vitro, the suspension spermatogenic cells which were round or oval and the adherent sertoli cells that were fusiform or irregular shape were both oberved in 32℃group and 37℃group. First 3 days, the number of germ cells and the state of growth were no significant different. Starting from the 4th day, the number of germ cells and the state of growth was gradually decreased in 37℃group, at the 4th day, the number of germ cells was 3.11±0.18x106/ml in 37℃group,which was 3.40±0.25×106/ml in 32℃group, at the 8th day, the number of germ cells was 0.49±0.25x106/ml,which was lower than 2.93±0.26 x 106/ml in 32℃group,the difference was significant (P<0.05). At 8th day cocultured in vitro, type A spermatogonias were successly separated by gradient centrifugation combined with differential adhesion method, the result of flow cytometry showed that the proportion of S phase in 32℃group was 49.35±1.04, which was significantly higher than 43.58±0.78 in 37℃group, the proportion of G0/G1 phase was 40.99±0.76, which was significantly lower than 46.45±0.92 in 37℃group, the difference was significant (P<0.05). the result of RT-PCR and Western Blot showed that the expressions of c-kit、PI3K and cyclinD3 mRNA and protein level in type A spermatogonias had been detected and in 32℃group were significantly higher than those in 37℃group (P<0.05). The gene sequencing results showed that the exon 9,11 and 13 of c-kit were no mutation in in 32℃group. In 37℃group, no mutation was detected in exon 11 and 13, while the peak was found in exon 9, suggesting deletion or insertion mutation may have been around exon 9 region.Conclusion:In basal medium of DMEM/F12 containing FBS body cavity temperature (37℃) influenced the proliferation of rat spermatogonia cultured in vitro and might lead to the mutation of c-kit gene, this temperature should be not as culturing temperature on studying normal proliferation and differentiation of spermatogonial cells in vitro.