Effects Of BMP4 And RA On The Differentiation Of Goat Spermatogonial Stem Cells In Vitro

Spermatogonial stem cells(SSCs)are a type of adult stem cells located in the seminiferous tubule basement membrane with self-renewal and differentiation capabilities,which are only adult stem cell in the body capable of transmitting genetic information.SSCs are the basis of spermatogenesis,which not only ensures the continuous production of sperm,but also the reproductive basis of the male animal during its life.It plays a very important role in breeding offspring.However,the SSCs system of differentiation has not been established,and the differentiation mechanism of SSCs from diploid cells to haploid sperm is not clear,all of this need further research.In this experiment,the effects of RA and BMP4 as two exogenous induced differentiation factors on goat SSCsdifferentiation in vitro.Firstly,SSCs were obtained by mechanical separation,two-step enzymatic method,and differential attachment.Secondly,exploring the optimal concentration and action time of SSCs induced by RA and BMP4,the SSCs in vitro differentiation system was constructed;Western blotting was used to detect the effects of BMP4 on Smad and PI3 K signaling pathways,and real time PCR was used to detect the effects of BMP4 on transcription factors.Finally,real time PCR was used to detect the effects of RA on BMP4 genes and receptors,Western blotting was used to detect Smad signaling pathway and P16,P21 in RA+BMP4 group.The main research results in this experiment are as follows:1.Goat SSCs were purified by two-step enzyme digestion and 2 h?2 h?12 h differential attachment method used in this study.The immunofluorescence staining results showed that purified SSCs can express a large number of molecules Maker CD90 and PLZF,indicating that the purified cells has SSCs activity.2.Real time PCR results show that: compared with other concentrations and exposure time,the best effect of regulating the differentiation of SSCs can be achieved by adding 50 ng / m L BMP4 alone for 24 h or 10 n M RA for 18 h(P<0.05).3.Western blotting showed that the relative expression of Smad and P-Smad proteins in BMP4 group was 1.4 and 1.2 after adding 50ng/m L BMP4 to the culture medium,which was significantly higher than other test groups(P>0.05).The relative expression levels of P-PI3 K protein were 0.7 and 0.65,which were significantly lower than those of other groups(P>0.05).Real time PCR results showed that the relative expression levels of ID2 and ID3 in the BMP4 group downstream of the Smad pathway were 1.6 and 1.9,which were significantly higher than those in the other experimental groups(P<0.05).The relative expression of the downstream gene PI3 K pathway Etv5 in the BMP4 group was 0.6,which was significantly lower than that(P<0.05).The expression level of the downstream gene Bc16 b of the PI3 K pathway in the BMP4 group was slightly lower than that in the control group and the BMP4 + Noggin group,but there was no significant difference(P>0.05).4.Real time PCR showed that the relative expression levels of transcription factors Sohlh2 and Ngn3 in the BMP4 group were 1.3 and 1.2,which were significantly higher than other groups(P<0.05).The expression of FGF2 was slightly higher in the BMP4 + Noggin group than in the other groups,but there was no significant difference(P<0.05).The expression of Pou3f1 in the BMP4 group was lower than that in the other groups and there was no significant difference(P>0.05).5.Real time PCR showed that the expression of BMP4 gene in RA group was significantly higher than that in control group at 12 h and 24h(P<0.05).The expression level of BMP4 receptor gene ALK3 was significantly higher in the RA group than in the control group(P<0.05),and the expression level of ALK6 at 12 h.Although the test group was higher than the control group at 24 h,there was no significant difference(P>0.05).6.Western blotting showed that the expression of P-Smad in RA + BMP4 group was significantly higher than that in RA + Noggin group,BMP4 group and Control group(P<0.05);the expression of P21 in the test group was significantly higher than other groups Group(P>0.05),the expression of P16 in RA + BMP4 group was higher than other groups but the difference was not significant(P>0.05).In summary,the addition of RA and BMP4 alone or in combination is beneficial to promote the differentiation of SSCs.BMP4 mainly induces differentiation through the Smad,PI3 K signaling pathway and the regulation of transcription factor expression.RA can induce differentiation through the Smad signaling pathway by promoting the expression of BMP4.