| Sertoli cell (SC) provides a variety of nutrition for spermatogenic cell in the process of differentiation and maturity, such as hormone, lipid, vitamin, et al; it is the Sertoli cell and spermatogenic cell that make up the main organizational structure of the testis’contorted seminiferous tubules. One of the most important conditions for the differentiation of spermatogenic cell lies in constant proportion of quantity of the SC and spermatogenic cell, the sperm production of adult animal testicles is decided by the number of SC. SC proliferation is regulated by multiple elements, while Skp2is the crucial one among all the hormone and factors in regulating cell proliferation, Skp2can be combined with polyubiquitinated p27kip1, which will prompt the degrade of p27kip1on protease of26S and contribute to cell proliferation.A large number of researches showed that estrogen plays an important part in proliferation of Sertoli cells, and PI3K/Akt get involved in regulating cell proliferation. The study on the mechanism of PI3K/Akt performed by estrogen helps to illustrate the mechanism of proliferation of Sertoli cell. The purpose of this research is to identify whether P13K/Akt regulate the expression of Skp2protein, Skp2protein, Skp2mRNA, CyclinEl mRNA, and CyclinDl mRNA in Sertoli cell of immature piglets.The present study was performed by culturing the immature boar sertoli cells as material and by the addition of estrogen receptor inhibitor and various signal pathways inhibitors, applying flow cytometry to test the change of the cell cycle and western blot to test the protein content of Skp2, p27kip1, PCNA; and real-time fluorescence quantitative PCR to test the relative amount expression of Skp2mRNA, CyclinEl mRNA, CyclinDl mRNA.The experimental results are stated as follows:(1) The concentration of10-9mol/L of17β-estradiol is the best in promoting the transform of SC from G1phase to S phase.(2)17(3-estradiol (10-9mol/L) promotes the activity of PI3K, Akt by the method of time-dependent, which reaches a peak in the30th minute (P<0.05).(3) G-15, the GPR30receptor inhibitors, inhibits the expression of PI3K which is induced by17β-estradiol (P<0.05), and receptor agonists G-1promotes the expression of PI3K (P<0.05).(4) PP2, the SRC inhibitor, inhibits the expression of PI3K which is induced by17β-estradiol (P<0.05).(5) LY294002, the PI3K inhibitor, inhibits the expression of Akt induced byl7β-estradiol(P<0.05).(6)17β-estradiol (10-9mol/L) promotes the expression of Skp2protein, PCNA protein, CyclinDl mRNA and CyclinEl mRNA (P<0.05), while it can reduce the protein expression of p27Kipl. LY294002as well as Akt inhibitors10-DEBC inhibits the activity of17β-estradiol (10-9mol/L)(P<0.05), yet there is no significant difference as the two inhibitors alone to act on the expression and blank comparison of Skp2protein, CyclinDl mRNA and CyclinEl mRNA (P>0.05).(7) LY29002and10-DEBC can reduce the transform of SC from G1phase to S phase,which is induced by17β-estradiol.In conclusion,17β-estradiol has an effect on the activity of P13K/Akt through GPR30, SRC, regulates Skp2proteins and the level of mRNA and CyclinDl mRNA, CyclinEl mRNA, promotes the transform of SC from G1phase to S phase, and then regulates the proliferation of SC. |
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