Expression Of GDNF In LPS-treated Mouse Sertoli Cells And Its Effects On Spermatogonial Stem Cells

Objective and Significance Orchitis is a common nonspecific infection of the male reproductive system, it can affect the sperm count and quality, and can cause infertility in severe cases. Lipopolysaccharide(LPS), which also called endotoxin, is the main component of gram-negative bacteria cell wall, and also is the important medium which can cause inflammation of testis cells when orchitis occurs., Intraperitoneal injection can build the orchitis model in rats. Revealing the mechansim of orchitis infertility caused by LPS has high value in clinical application.Spermatogonial Stem cells(SSCs), who located on the basement membrane of testicular seminiferous tubules, are the only adult stem cells who can transfer the genetic information to the daughter cells in natural state. It can through self-renewal to maintain the stability of its own number, and can through a series of differentiation to produce spermatozoa eventually. The balance between self-renewal and differentiation of SSCs ensure ongoing of spermatogenes in the whole life of male, Once the balance is broken could lead to spermatogenetic dysfunction:Excessive proliferation can lead to germ cell tumor, while excessive differentiation can lead to male infertility.Sertoli cells(SCs) located on the endothelium of testicular seminiferous tubules, they were discoved and described by Entico Sertoli, who was a Germanic histologist. It provides critical support and nutritional function to SSCs. Glial cell-line derived neurotrphic factor(GDNF), which is produced by SCs, is the most important cell factor for the self-renewal of SSCs, while the stem cell factor (SCF)、bone morphogenesis protein4(BMP4) are important cell factor produced by SCs for the differentiation of SSCs. At present, evidence has showed that LPS can cause expression of various cell factors change in SCs, But little is known about LPS-treated SCs could on the SSCs, and the mechansim is also unknown.In our study, we culture mouse SCs and SSCs in vitro, Immunofluorescent stainings and RT-PCR were performed to investigate the LPS-treated SCs’s effects on the self-renewal and differentiation of the SSCs, RT-PCR and Western blot were performed to investigate the reasons of these effects. The mechansim of orchitis infertility caused by LPS was revealed, a new experimental basis for the treatment of male infertility was provided.Methods1. SCs culture in vitro and treatment in groups Testes from5～6weeks old KM mice were extracted and digested by two-step enzymatic method, differential attachment method were performed to purify SCs, then divided the cells into four groups:the normal culture medium(Con group), the medium with LPS treat1day (1d group)、treat2days(2d group) and treat3days (3d group), the final concentration of LPS in each medium were10μg/mL. After the treatment, the medium with LPS was replaced by LPS-free fresh SSCs medium to continue the culture for48h, then the medium of the four groups were collected respectively as conditioned medium and mixed at the ratio of1:1with GDNF-free fresh SSCs medium to culture the SSCs, while the groups of SCs were reserved. 2. SSCs culture in vitro and treatment in groups Testes from5-7days old KM mice were extracted and digested by two-step enzymatic method, differential attachment method were performed to purify SSCs, then divided the cells into four groups:con, group I, group II, group III and cultured for96h in the contidioned medium respectively having been prepared, then collected the groups of cells, immunofluorescent stainings were performed on SSCs to investigate the expressions of PLZF、oct4、 PCNA、and c-kit、sohlh2; RT-PCR was also performed on SSCs to investigate the expressions of Bc16b and Etv5.3. RT-PCR was performed on the groups of SCs to investigate the expressions of GDNF、PCNA and BMP4, Western blot was also performed on the groups of SCs to investigate the expressions of GDNF.Results1. SSCs immunofluorescent stainings results revealed that the expression of PLZF and PCNA was decreased in the order of group Con, group Ⅰ, group Ⅱ, group Ⅲ, the highest was the group Con and the lowest is the group Ⅲ, the differences between treated groups and Con group have statistical significance(P<0.01); While the expression of c-kit and sohlh2was increased in the order of group Con, group Ⅰ, groupⅡ, group Ⅲ, the highest was the group Ⅲ and the lowest is the group Con, the differences between treated groups and Con group have statistical significance(P <0.01).2. SSCs RT-PCR results revealed that the mRNA level of PLZF and oct4was decreased in the order of group Con, group Ⅰ, groupⅡ, group Ⅲ, the differences between treated groups and Con group have statistical significance(P<0.01); While the mRNA level of c-kit and sohlh2was increased in the order of group Con, group Ⅰ, groupⅡ, group Ⅲ, the differences between treated groups and Con group have statistical significance(P<0.01). 3. SSCs RT-PCR results revealed that the mRNA level of Bcl6b and Etv5was decreased in the order of group Con, group Ⅰ, group Ⅱ, group Ⅲ, the differences between treated groups and Con group have statistical significance (P<0.01).4. SCs RT-PCR results revealed that the expression of GDNF in SCs was decreased in the order of group Con, group Id, group2d, group3d, the differences between treated groups and Con group have statistical significance (P<0.01). While the expression of SCF and BMP4between groups and Con group have no statistical significance(P>0.05).5. SCs western blot results revealed that the GDNF protein was decreased in the order of group Con, group Id, group2d, group3d, the differences between treated groups and Con group have statistical significance(P <0.01).Conclusion1. The differentiation of SSCs can be stimulated and the self-renewal of SSCs can be inhibited respectively by the LPS-treated SCs.2. The expression of GDNF in LPS-treated mouse SCs is decreased, this change can be inhibited the self-renewal of SSCs via down-regulation of Bcl6b and Etv5expression.