The Mechanism Of GDNF Mediates Self-renewal Of Dairy Goat Male Germ-line Stem Cells

Male germline stem cells(mGSCs), is one of the most active and attractive biological research areas in recent years.The study on mGSCs has a broad application prospects in the field of medicine and biology. Glial cell line-derived neurotrophic factor(GDNF) was the first found growth factor which could regulate self-renewal of mGSCs. GDNF is the first molecule known to regulate the cell fate decision of SSC and data also show that GDNF mediates the self-renewal and differentiation of undifferentiated spermatogonia in a dose-dependent manner.Researches on mice have demonstrated that the regulation of GDNF on SSCs mainly through Ras/Erk1/2, Src family kinase, PI3K/Akt signaling pathway, but for different species, different cell lines, the signaling pathways GDNF-mediated are different from each other. Meanwhile, whether GDNF could regulat the self-renewal of SSCs isolated from cattle or goats have not been reported.In this research, we first examined expression patterns of GDNF family receptor GFRa1 in different ages of dairy goat testises. And then, the purified mGSCs were isolated from dairy goat testis tissue using mixed enzymes and fibronectin. Immunocytochemical staining revealed that the isolated and purified cells express GFRa1,CD49f,Stra8,Vasa,TERT,Oct4, reflecting that these cells are phenotypically mGSCs. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL) and found that 10 ng/mL was the optimal concentration to maintain self-renewal of dairy goat mGSCs. Finally, we cultured dairy goat mGSCs in mediums contains GDNF (10 ng/mL) or supplemented with inhibitor of PI3K or MEK respectively, and found the main signaling pathway GDNF-mediated.1. Expression pattern of GDNF family receptor GFRa1Expressions of GDNF family receptor GFRa1 in the fetal, neonatal, adolescent and adult dairy goat testis tissues were detected by RT-PCR and immunohistochemical. RT-PCR results showed that, GFRa1 relatively strongly expressed in the fetal period of dairy goat testis and the expression level was gradually reduced with the proliferation of the dairy goat's age. Immunohistochemistry results showed that GFRa1 only expressed in the adult testis. These results indicated GDNF family receptor GFRa1 expressed in dairy goat testis, but the expression pattern may be quite different from that in mouse and human testis. 2. Isolation, purifition and identifition of dairy goat mGSCsDairy goat testicular tissue was digested using mixed enzymes (collagenase 2 mg/ml, DNase 20μg/ml, Dispase 2 mg/ml) and then cells adhered in or not in 2 hours were identified by AP staining. It showed that cells did not adhered in 2 hours appeared whole clusters stained and the cells adhered in 2 hours showed partly AP positive of the cluters which meant that cells did not adhered in 2 hours contain more mGSCs. Immunocytochemical staining of the cells did not adhered in 2 hours revealed that these cells expressed GFRa1,CD49f,Stra8,Vasa,TERT,Oct4, reflecting that we obtained puified dairy goat mGSCs.3. Optimal concentration of GDNF on dairy goat mGSCsIsolated and purified dairy goat mGSCs were cultured in mediums supplemented with different concentration of GDNF (0 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL). By comparing the population doubling time (PDT), capacity of cell proliferation (immunofluorescence staining by PCNA and BrdU ) and pluripotency of these cells we found that medium supplemented with 10 ng/ml GDNF was more effective to maitain self-renewal of dairy goat mGSCs.4. Signal pathway mediated by GDNFIsolated and purified dairy goat mGSCs were cultured in control medium or mediums contain 10 ng/ml GDNF or 10 ng/ml GDNF supplemented with inhibitor of PI3K (10μM ) or MEK(10μM). The mGSCs cultured in different mediums were compared through the population doubling time (PDT), capacity of cell proliferation (immunofluorescence staining by PCNA and BrdU ) , pluripotency, RT-PCR, Real time PCR, Western blotting and flow cytometry. Results showed that GDNF maintains self-renewal of dairy goat mGSCs through PI3K/Akt and MEK/ERK1/2 signal pathway and it also seemed that GDNF mainly through PI3K/Akt pathway to maintain self-renewal of dairy goat mGSCs.