| Sertoli cell is a very important function cell in contorted seminiferous tubules epithelium, it can provide hormones, lipids, vitamins and many other nutrients for differentiation and maturation of germ cells; and constitute main organization structure of testis' contorted seminiferous tubules with spermatogenic cell. The function of sertoli cell is regelated by the hypothalamus-pituitary-gonadal axis endocrine, the blood-testis barrier can well protect germ cells. The number of sertoli cells protect germ cells is limited, so the number of sertoli cells determines the number of testicular sperm in a certain degree.FSH can promote the proliferation of sertoli cells by dose dependent manner in 0-50ng/ml, but when the concentration of FSH is too high, the role in promoting proliferation decreases; 17β-estradiol can promote the proliferation of immature sertoli cells in rats or pigs in the culture conditions, and they are associated with cAMP, ERK1/2 signaling pathway, but when FSH and 17β-estradiol was combined, it is not clear how they regulate expression of S-Phase Kinase-Associated Proteins2(Skp2) in cultured immature boar Sertoli eel. So, in the study, cultured immature boar Sertoli cells were used as material, while a variety of signaling pathway inhibitors were added, Western blottingting and Real-time RT-PCR were used to detect the expression of Skp2 protein and mRNA to clarify what signaling pathways to regulate the expression of Skp2 protein and mRNA when Combined FSH and 17β-estradiol. to lay the foundation for the further understanding of the molecular mechanism of Sertoli cells proliferation.In the experiment, Immature Boar is the research object, treatment of combine FSH and 17β-estradiol on Sertoli cell, after Omin,15min,30min,60min,90min, Sertoli cells are collected for extracting protein and mRNA, Western blottingting were used to detect the expression of Skp2 protein, and Real-time RT-PCR were used to detect the expression of Skp2 mRNA. It was found, FSH and 17β-estradiol promotes expression of Skp2 protein, the amount of protein is largest in 30min, after, the expression of Skp2 gets down, in 90min, expression of Skp2 protein is no difference compared with the control; after 15min of effect of combine FSH and 17β-estradiol on Sertoli cell, the expression of Skp2 mRNA increases 1.93 times (P<0.05), in 30 min, the effect is largest, compare with control, the expression of Skp2 mRNA increases 7.39 times (P<0.05), then the expression of Skp2 mRNA decreases, in 90 min, compare with control, the expression of Skp2 mRNA increases 3.06 times(P<0.05). Experiments show that combined FSH and 17β-estradiol can promote the expression of Skp2 protein and mRNA.The further experiment study the effect of cAMP,PKA,Ca2+ and ERK to FSH and 17β-estradiol 1 induced the expression of Skp2. Addin the Forskolin which is the accelerant of cAMP promote the expression of Skp2 protein, but the dose of protein is lower than FSH and 17β-estradiol. Addin Rp-cAMP(cAMP inhibitor), H-89(Protein Kinase A inhibitor), verapamil (L-type Ca2+ ionic channel inhibitor)and U0126(ERK1/2 inhibitor) respectively. They inhibit FSH and 17beta-estradiol induced the expression of Skp2 protein; and Forskolin promote the expression of Skp2 mRNA obviously, compare with control, Skp2 mRNA increase 8.98 times (P<0.05), but lower than the FSH and 17β-estradiol。Rp-cAMP,H-89,Verapamil,U0126 all affect FSH and 17β-estradiol 1 induced the expression of Skp2 mRNA, compare with FSH and 17beta-estradiol, the expression level of Skp2 mRNA decreases 50.66%,53.96%,51.46%,57.97% respectively (P<0.05)The experiment also analyzed the Effect of PKA and Ca2+ on the activity of ERK1/2 in Sertoli cells. The results showed that compared with the control, FSH and 17β-estradiol enhances the activity of ERK 1/2, but the effect had no significant difference with FSH and 17β-estradiol alone. Compare with the control, H-89,Verapami alone had no significant effect on the activity of ERK 1/2, but they reduced the activity of ERK 1/2 with FSH(50ng/ml) and 17beta-estradio(10-9 mol L-1).Conclusions:Combined FSH and 17β-estradiol activate cAMP-PKA pathway and Ca2+ influx, and both of them influence ERK1/2 activation,while ERK1/2 enhances the expression of Skp2.
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