A Preliminary Study On The Correlation Of DDIT3-TRIB3-AKT Signal Pathway In Spermatogenesis

Objective To construct the co-culture system of rat testicular tissue cells in vitro and verify the existence of DDIT3-TRIB3-AKT signal pathway in rat testis.Methods(1)To establish a co-culture system of rat testicular tissue cells in vitro: rat testicular tissue was taken and cultured in DMEM/F12 medium containing 12% fetal bovine serum(FBS)under aseptic condition,and the culture medium was changed every other day;(2)Identification of co-culture system of rat testicular tissue cells in vitro: using co-cultured cells as materials,total RNA,was extracted and reverse transcribed into c DNA,to identify the expression of marker genes in different stages of spermatogenesis by Real-time q PCR;(3)To detect the expression of DDIT3-TRIB3-AKT signaling pathway related proteins and m RNA: using co-cultured cells as materials,total protein,Western blot was extracted to detect the expression levels of DDIT3,TRIB3 and AKT proteins.Total RNA,was extracted and reverse transcribed into c DNA,Real-time q PCR to detect the m RNA expression of DDIT3,TRIB3 and AKT genes;(4)The expression of signal pathway related proteins was detected by Western blot after inhibition of DDIT3 and TRIB3 genes by small interfering RNA technology:the expression of DDIT3,TRIB3 and AKT proteins was detected by Western blot after inhibition of DDIT3.The expression of DDIT3,TRIB3 and AKT protein was detected by Western blot after TRIB3 was inhibited;(5)The m RNA expression level of signal pathway related factors was detected by Real-time q PCR after suppression of DDIT3 and TRIB3 genes by small interfering RNA technology:the m RNA expression of DDIT3,TRIB3 and AKT genes was detected by Real-time q PCR after suppression of DDIT3.The m RNA expression of DDIT3,TRIB3 and AKT gene was detected by Real-time q PCR after TRIB3 inhibition.Results(1)The co-culture system of rat testicular tissue cells in vitro was established successfully.In this culture system,spermatogonial stem cells,spermatocytes,round sperm cells and Sertoli cells could coexist for a long time in vitro for 12 weeks;(2)After 6 weeks of culture,Real-time q PCR detected marker genes CDH1,SYCP3 and TP2 at different stages of spermatogenesis;(3)The expression of DDIT3,TRIB3 and AKT protein was detected by Western blot,and the m RNA expression of DDIT3,TRIB3 and AKT gene was detected by Real-time q PCR in the co-culture system;(4)The results of Western blot and Real-time q PCR showed that TRIB3 was the downstream molecule of DDIT3 and the upstream molecule of AKT,after inhibition of DDIT3,TRIB3 expression decreased and AKT expression increased.Conclusion(1)Germ cells in the co-culture system can continue spermatogenesis in vitro under the nourishment of tissue and Sertoli cells,so this system can be used to verify the signal pathway related to spermatogenesis in vitro;(2)DDIT3-TRIB3-AKT signal pathway exists in rat spermatogenesis.