Research On The Effect Of NO On Cytoskeleton In Sertoli Cells

Nitric oxide is an information element which has a dual role in male reproductive.Sertoli cells'(SC) integral skeleton structure were the basises for maintaining spermatogenesis.A variety of drugs into the testis may produce excessive NO,which play role on sertoli cells primary.To study the impact and mechanisms that NO play on SC cytoskeleton is not only to further understand the regulatory mechanism of spermatogenesis,to enrich and improve the mechanisms of developmental biology in male gonad,but also to lay the foundation for the treatment of male sterility and to provide a basis for drug safety evaluation.To set up a suitable experimental model,we used piglets for cultivating SC in vitro and treated SC with sodium nitroprusside(SNP) which could produce NO and analog cellular environment.We observated the impact of NO on the skeleton structure of SC and the secretion of transferrin. Furthermore,we also detected the antioxidant level of SC and the activity of p38MAPK(p38 mitogen-activated protein kinase).First of all,we used 1μmol/L,10μmol/L,100μmol/L and 1 mmol/L SNP to deal with SC for 24 h.The results showed that as the concentration of SNP increased,the level of NO within SC significantly increased.The level of NO within SC was between 0.07±0.01μmol/L and 2.74±0.03μmol/L,which not only covered the normal physiological concentration but also included the concentrations higher than 1μmol/L.Meanwhile we ruled out the effect of endogenous NO on this experiment.Moreover,we detected the impact of different concentrations of SNP on cell viability of SC.Compared to controls,cell viability in the group of 1μmol/L SNP had increased by about 15.90 %,respectively(P＜0.05).As the concentrations of SNP increased,cell viability decreased. Compared to controls,when the concentrations of SNP were 10μmol/L,100μmol/L and 1 mmol/L, cell viability had separately increased by about 2.55%,12.36%and 59.89%,respectively(P＜0.05).Cultivating SC in vitro for 24h,we added culture medium with 1μmol/L,10μmol/L,100μmol/L and 1 mmol/L SNP.After cultivating SC,we collected cell protein extraction and quantificated protein.The results inferred that high concentrations of NO could improve the activity of p38MAPK in SC by western blot.Compared to controls,the activity of p38MAPK in the group of 1μmol/L,10μmol/L,100μmol/L and 1 mmol/L SNP had separately increased by about 15.38%, 16.92%,36.92%and 69.23%,respectively(P＜0.05).Than,cell immunofluorescence test results showed that thin and dense microfilament distributed in the cytoplasm more evenly with clear reticular formation in the group of 1μmol/L,10μmol/L,100μmol/L SNP and 1 mmol/L SNP plus SB203580 as well as controls.However,at 1 mmol/L,SNP have destroyed the actin stress fiber,micro-tow and set microfilaments to the brink of aggregation.We also found thatα-tubulin radically arranged around the nucleus as well as controls, when SC was added to 1μmol/L,10μmol/L,100μmol/L SNP and 1 mmol/L SNP plus SB203580. But when the concentration of SNP was 1mmol/L,microtubule net completely damaged with dispersion-shaped.Furthermore,the expression of transferrin decreased as the concentration of SNP increased. When the concentration of SNP was 1μmol/L and 10μmol/L,the expression of transferrin was similar to controls(P＞0.05).But compared to controls,the expression oftransferrin in the group of 100μmol/L and 1 mmol/L SNP had separately decreased by about 15.79%and 25.26%,respectively (P＜0.05).And compared to the group of 1 mmol/L SNP,When SC was treated with 1 mmol/L SNP plus SB203580,the expression of transferring had increased by 30.99%,respectively(P＜0.05).This results indicated that NO not only undermined the cytoskeleton,but also reduced the secretory function of cell.We also found that high level NO could increase the activity of p38MAPK and decrease the activity of SOD,GSH and T-AOC in Sertoli cells.On the whole,we got these conclusion:(1) The model is applicable to set up research on the impact of NO on the function of SC.(2) Low concentration of NO could guarantee the survival of cells,while the high concentration of NO had reduced cell survival.(3) SNP resulted in the increase of NO in sertoli cells,and decreased antioxygen ability.The high level NO destroyed cells' microfilament and microtubule through activating p38MAPK,thus decreased cell secretion.