Preliminary Study On BMP4 Regulating Testicular Sertoli Cell Proliferation

Spermatogenesis and healthy testicular development are prerequisites for male reproductive function.The entire process of spermatogenesis is closely related to the regulation of surrounding sertoli cells,testicular sertoli cells can directly interact with germ cells in the spermatogenic tubules,or nourish germ cells by secreting products to regulate the synchronous development of germ cells and then regulate spermatogenesis.The function study how the regulators affect sertoli cells and their physiological functions.As a member of the TGF-TGF family,BMP4 plays an important role in embryonic development and cell proliferation and differentiation.Studies have shown that Id2 gene can be used as a downstream gene of BMP4 to maintain the dryness of embryonic stem cells and is related to cell proliferation.Through transcriptome sequencing analysis of goat testicular tissue,we found that BMP4 still has a targeted relationship with Id2 gene.However,whether Id2 can participate in BMP4-induced cell function change in goat testicular sertoli cells and its specific regulatory mode remains to be further studied.In this paper,testicular sertoli cells of dazu black goat were taken as the research object.In vitro culture,immunofluorescence,BMP4 interference,overexpression,q PCR and Western Blot were used to verify whether BMP4 regulated the testicular sertoli cells of goat through Id2 and their regulatory relationship.The following results are obtained:1.Through collagenase and trypsin two-step digestion method,the Sertoli cells of goat testis were separated.the cells were purified by differential adherence method,and the sertoli cells were identified and viability detected by HE,forgen and immunofluorescence.The purity of sertoli cells reached over 98%.2.BMP4 was detected to be expressed in sertoli cells at different ages in goats,and was significantly higher at 2 months than at 0,1 and 3 months(p <0.01),indicating that BMP4 may play a certain role in testicular development during puberty.3.BMP4 protein can promote the proliferation of goat testicular cells,and the higher the concentration,the more obvious the proliferation effect.Among them,the testicular sertoli cells proliferated the most when the concentration of BMP4 was 200ng/m L and the culture time was 72 h.4.After interfering with BMP4,the gene levels of PCNA and Id2 and the expression levels of Id2 protein were significantly reduced(p <0.05).Compared with the interference group,the gene expression level of PCNA in the untreated control group was reduced by 42%,with significant difference(p<0.01).The gene expression level of Id2 was decreased compared with the control group,but there was no significant difference(p>0.05).However,the protein level of the control group was decreased by39.2%,with significant difference(p<0.05).The results showed that after interfering with BMP4,the proliferation of sertoli cells was limited and Id2 could be regulated.5.After the interference of BMP4,the proliferation cycle was detected by flow cytometry,It was found that the proliferation index of the interference group was significantly decreased compared with the NC group(p<0.05).6.After the overexpression of BMP4,the gene expression of Id2 was 1.25 times that of the control group(p<0.05).Compared with the control group,the protein level of Id2 increased by 18%(p<0.05).It shows that BMP4 has positive regulation effect on Id2.7.In the Id2-interfering group,the expression levels of PCNA gene and protein were significantly reduced compared with the overexpression of BMP4 and the interference with Id2 group(p<0.05),further proving that BMP4 can regulate Id2 gene and protein levels to affect the proliferation of sertoli cells.8.After BMP4 overexpression,the phosphorylation level of P38 MAPK protein significantly increased compared with the control group,42% higher than the control group(p<0.05),indicating that BMP4 overexpression can also activate the P38 MAPK protein pathway.In summary,BMP4 can promote the proliferation of Goat testis sertoli cells by upregulation of Id2 gene and protein levels.This study provides a basis for elucidate the molecular mechanism and physiological function of BMP4 on the sertoli cells of goat testis.