| Purpose: To detect the expression of B7-H3 in mouse testis at different ages and its effect on the proliferation of mouse spermatogonial stem cells,and to explore the underlying molecular mechanism.Methods: This study consists of three parts.1.Immunohistochemistry and western blot were used to detect the expression of B7-H3 in C57BL/6 mouse testis at different age(3 weeks,8 weeks,4 months,9 months).Then,we analyzed the association between the expression of B7-H3 and the age of mice.2.We extracted mouse spermatogonial stem cells from 1 week old C57BL/6 male mice testis,and identified them.CCK8 was used to detect the proliferation of mouse spermatogonial stem cells,which were cultured with different concentration of B7-H3(0,5,10,25ng/ml)for different time(1,6,12,24,48,72 h)in vitro.In addition,BRDU was used to determine the effects of B7-H3(0,5,10,25ng/ml)on the cell cycle of spermatogonial stem cells that were cultured for 48 and 72 hours.3.Western blot was used to explore the expression of phosphorylation of PI3 K,ERK1/2 and JNK1/2/3 in mouse spermatogonial stem cells that were cultured with 10ng/ml B7-H3 for different time(0,15,30,60 min),and the levels of phosphorylation of PI3 K expressed in mouse spermatogonial stem cells that were cultured with B7-H3(0,5,10,25ng/mL)for 30 minutes.Furthermore,BRDU was used to detect changes of the cell cycle of mouse spermatogonial stem cells,which was treated with 10 uM LY294002 before incubation with 10ng/ml B7-H3.Results:1.B7-H3 was mainly expressed in the membrane and cytoplasm of mouse sertoli cell.The level of B7-H3 expressed in the testes of 4 months old mouse was the highest(all P <0.01).Compared with 4 months old mouse,the levels of B7-H3 expressed in mice at the age of 3 weeks,8 weeks and 9 months were 22.76%±1.31%,64.40%±6.25% and 65.36%±4.20%.These results indicated that the expression of B7-H3 in mouse testis changed with the development of sexual maturation.2.The expression of C-KIT was low on the surface of the extracted cells,while OCT-4 was highly expressed,which were consistent with the characteristics of mouse spermatogonial stem cells.Moreover,the results of CCK8 showed that regardless of the concentration of B7-H3,from 1 to 48 hours,the proliferation rate of mouse spermatogonial stem cells increased;however,from 48 to 72 hours,the proliferation rate of mouse spermatogonial stem cells decreased.After 48 hours of incubation,the proliferation rates of mouse spermatogonial stem cells treated with 10 ng/ml(OD value,1.25±0.04)and 25 ng/ml(OD value,1.21 ±0.06)B7-H3 were both significantly higher than those of mouse spermatogonial stem cells treated with 0 ng/ml(OD value,1.08±0.02)and 5 ng/ml(OD value,1.07±0.06)B7-H3(all P<0.001).There was no significant difference between the other groups(all P>0.05).And the proliferation rate of mouse spermatogonial stem cells were cultured for 72 hours was similar to the cells cultured for 48 hours.The results of BRDU showed that after 48 hours of incubation,the percentage of mouse spermatogonial stem cells in S+G2/M phase(cell proliferation index)was significantly higher in the 10 ng/ml(89.20%±2.07%)and 25 ng/ml(89.00%±1.61%)B7-H3-treated groups than that in the groups treated with 0 ng/ml(72.77%±1.53%)and 5 ng/ml(77.43%±4.01%)B7-H3(all P<0.001).There was no significant difference between the other groups(all P>0.05).However,after 72 hours of incubation,only the 10 ng/ml B7-H3-treated mouse spermatogonial stem cells had a higher S+G2/M percentage compared to that of cells treated with 0 ng/ml B7-H3(P<0.05).There was no significant difference between the other groups(all P>0.05).These results showed that B7-H3 could promote the proliferation of mouse spermatogonial stem cells.3.Cultured with 10 ng/ml B7-H3 for 30 minutes,the expression of phosphorylation of PI3 K in mouse spermatogonial stem cells was significantly higher than that in the cells were cultured for 0,15,60 minutes.However,there were no significant changes in phosphorylation of ERK1/2 and JNK1/2/3 after treatment with B7-H3 for different times(all P>0.05).Besides,the 10 ng/ml and 25 ng/ml B7-H3-treated mouse spermatogonial stem cells had higher phosphorylation of PI3 K than that of cells treated with 0 ng/ml and 5 ng/ml B7-H3(all P<0.0001),but there was no significant difference between 10 ng/ml and 25 ng/ml B7-H3-treated groups(P>0.05).Moreover,the percentage of S+G2/M phase in mouse spermatogonial stem cells treated with LY294002 and B7-H3 was significantly lower than that in the cells only treated with B7-H3(P<0.05).These results suggested that B7-H3 promoted the proliferation of mouse spermatogonial stem cell via PI3 K signaling pathway.Conclusion: B7-H3 was mainly expressed in mouse sertoli cells,and its expression was related to sexual maturation.In addition,B7-H3 promoted the proliferation of mouse spermatogonial stem cells by activating PI3 K signaling pathway. |
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