Effects Of Selenium On The In Vitro Proliferation And Apoptosis Of Sheep Spermatogonial Stem Cells

The purpose of this study was to investigate the effects of Se (from sodium selenite) on in vitro proliferation and aopotosis of sheep spermatogonial stem cells (SSCs).4-6 month Jinzhong sheep testes were collected for the present study. The SSCs isolated and purified using Percoll discontinuous gradient combined with differential plating were divided into five experimental groups that were cultured in culture media adding various concentrations of Se (0,2,4,8, or 16?mol/L). Each treatment was set up 5 duplications, and the medium was changed each 48 h. After 96 h, immunofluorescence was performed to identify and evaluate the proliferation of SSCs using a specific SSCs marker called promyelocytic leukaemia zinc-finger (PLZF). The methyl thiazolyl tetrazolium (MTT), cridine orange-ethidium bromide (AO/EB) and flow cytometric analysis were used to ditect the proliferation rate, viability and apoptosis rate. Further, the intracellular reactive oxygen species (ROS) level was measured at the 48 h,96 h, and the mRNA and protein expression of apoptosis-related genes (P53, Bax, Bcl-2, Caspase3/8) and cell cycle-related genes (CyclinB1?CDK1?p21?p27) were analyzed on 96 h using qRT-PCR and Western blotting. The results were as follows:(1) The single cell suspension was treated by percoll density gradient centrifugation and found that the SSCs were focused at the layer of 19%?35% Percoll, and its purity was up to 80% after further purification by attachment culture.(2) To evaluate the effect of Se on SSCs proliferation in vitro, the different doses of Se were added to the cell culture medium and cells were cultured for 96 h.The SSCs were in the undifferentiated state and Grapelike clusters of the SSCs colonies in all groups irrespective of Se concentration using immunofluorescence analysis with anti-PLZF antibody after 96 h of culture. The significant higher viability and proliferation rate of SSCs were observed in the treatment of Se with concentraion of 2?mol/L, compared to those from other groups (P<0.05). With the increase of Se addition, viability and proliferation rate were gradually declined, and cell apoptosis rate was obtained with the opposite trend compared to the viability and proliferation rate.(3)To examine the effects of addition of Se on intracellular ROS production, the relative intracellular ROS levels were estimated at 48 h and 96 h of culture. The highest and lowest intracellular levels of ROS were obtained in the 2?mol/L and 16?mol/L groups, respectively. With prolonged incubation time, intracellular ROS level for each Se group gradually increased.(4) To further investigate whether Se could improve the proliferation and apoptosis of sheep SSCs, the expression levels of cell cycle and apoptosis-relatation genes were examined using qRT-PCR and Western blotting. The mRNA expression of CyclinBl, CDK1 and Bcl-2 in the Se treatment groups were increased with the increasing of Se concentrations. But the opposite trend were obtained with the mRNA expression of p53. p21, p27, Box and Caspase3/8. The mRNA expressions of CyclinBl and Bcl-2 in the 2 ?mol/L group were significantly higher than that in other groups and p53, Box, Caspase3/8 were significantly lower than that in other groups (P<0.05). Although the mRNA expressions of CDK1,p21 and p27 in the 2?mol/L group were higher than that in the 4?mol/L group, no significant differences in those groups.The same trend protein expression of P53, P21, P27, CDK1 and Bax campared to mRNA expression, respectively.The present study showed that Se supplement at appropriate concentration (2?mol/L) to culture medium could promote the proliferation and excess Se exerted inhibitory effect on proliferation and induced apoptosis of sheep SSCs. It is the first to suggest this effect is likely to involve modulation ROS and affecting the cell cycle and aopotosis-related genes that arrests cell in vitro proliferation and apoptosis of sheep SSCs.