Effects Of PDGF-BB And EGF On The Proliferation Of Goat Spermatogonial Stem Cells In Vitro

Spermatogonial stem cells（SSCs）are special and unique diploid cells that can transmit the genetic material of mammals to their offsprings.At present,SSCs have been used for the production of transgenic animals and the recovery of male fertility.However,the proliferation technique of SSCs in vitro is still immature,and the mechanisms and pathways involved in the proliferation of SSCs are not yet clear.To investigate the effects of epidermal growth factor（EGF）and platelet-derived growth factor-BB（PDGF-BB）on goat SSCs proliferation in vitro,6 testis of 60-to 75-day-old goats were minced and digested using a modified two-step enzymatic digestion process.Then,goat SSCs were purified twice by differential plating,and were identified by alkaline phosphatase staining（AKP）and indirect immunofluorescence cell analysis.After that,different doses of PDGF-BB and/or EGF were supplemented into Dulbecco’s modified eagle medium（DMEM）.The absorbance of each group was measured by methylthiazolyl tetrazolium（MTT）assay at day 3,day 6 and day 9,respectively.Following the selection for optimum concentration of PDGF-BB and EGF,the protein expression levels both of ERK1/2 and phosphorylated-ERK1/2,and the expression of apoptosis-related genes（BCL2,BAX and BAD）of control group and three best experimental groups were tested and analysed using western blot and real time PCR,respectively.After SSCs were treated with specific MEK1/2 inhibitor PD0325901 for 6 days,the expression of above proteins and genes were detected again.The main results of this research were as following:1.High purity SSCs were obtained from testicular single-cell suspension after twice differential plating.The result of AKP assay showed that most of the isolated cells were AKP positive cells,and that the majority of round or oval cells was stained into black.And the results of immunofluorescence staining showed that the purified cells expressed the SSCs markers（THY1 and PLZF）.2.Compared with the control group,adding 25 ng/mL EGF or 35 ng/mL EGF significantly promoted the self-renewal of goat SSCs（P<0.05）,but the difference between them was not significant（P>0.05）.The effect of adding 20 ng/mL PDGF-BB alone on the proliferation of goat SSCs was significantly higher than that of the control and other experimental groups（P<0.05）.The combination of 20 ng/mL PDGF-BB and 25 ng/mL EGF strongly enhanced the in vitro proliferation of goat SSCs（P<0.05）,and the OD₄₉₀ value was0.736 at day 6.3.The results of western blotting showed that:in comparison with the control group,the expression of ERK1/2 protein of three experimental groups increased in varying degrees,with the largest increase in the combined group,and the smallest change in 20 ng/mL PDGF-BB group.After the specific MEK1/2 inhibitor was added,the expression of ERK1/2 protein of the four groups decreased,but the rangeability of the three experimental groups was less than that of the control group.The expression of phosphorylated-ERK1/2 of the control group was much lower than that of the three experimental groups,and the expression level of the latter was about 3 to 5 times as much as that of the former.After the inhibitor was added,the expression of phosphorylated-ERK1/2 protein of the four groups decreased to 10%to 30%of the original expression level.4.The results of real time PCR indicated that:compared with the control group,the expression of BCL2 was upregulated in the three experimental groups.And the BCL2expression of the 20 ng/mL PDGF-BB plus 25 ng/mL EGF group was significantly higher than other groups（P<0.05）.The BCL2 level in 25 ng/mL EGF group was significantly higher than that of the control（P<0.05）,however,the expression level of anti-apoptotic gene BCL2 in 20 ng/mL PDGF-BB group was not significant compared to the control group（P>0.05）.Comapred with all other experimental groups,the expression levels of apoptosis genes BAX and BAD in the control group were significantly increased（P<0.05）,while the differences between the experimental groups were not significant（P>0.05）.After PD0325901 was added,the expression of anti-apoptotic gene BCL2 in all treatment groups reduced significantly（P<0.001）,just about 10%²0%of the original level,while BAX and BAD expression levels were significantly higher（P<0.001）.Conclusion:both EGF and PDGF-BB were beneficial to the self-renewal of goat SSCs.We speculated that these two kinds of growth factors may through the Ras/ERK1/2 signaling pathway to regulate the expression of phosphorylated-ERK1/2 protein in goat SSCs,and thus enhanced the resistance of SSCs to apoptosis.