Cloning Of Yak Cyclin D1/ E1 Gene And The Effects Of IGF-I/TGF-?1 On Expression Of Cyclin D1 In Sertoli Cell

Cyclin D1 and Cyclin E1 are the pivotal protein of G1/S phase.The aim of this study was to clone the CDS of yak?Bos grunniens?Cyclin D1 and Cyclin E1 gene,to analyse their biological characteristics,and to research the effects of insulin-like growth factor-I?IGF-I?and transforming growth factor-?1?TGF-?1?on Cyclin D1 in Sertoli Cells?SCs?.The Cyclin D1 and Cyclin E1 gene were cloned by RT-PCR.The expression of Cyclin D1 and Cyclin E1 proteins in SCs were localized by immunofluorescence staining.The effects of the different concentration IGF-I(0?contral group?,25,50,100,150 ng?mL^-1),the different time?0?contral group?,5,12,18,24 h?and concentration(0?contral group?,250,500,750,1000 pg?mL^-1)TGF-?1on Cyclin D1 mRNA were detected by Real-time quantitative PCR?qRT-PCR?and that of protein by Western bloting.The results were as follows:?1?Yak Cyclin D1 and Cyclin E1gene had the high homology with other species,GenBank accession number were KY 420723 and KY437813 respectively.Their protein belonged to nuclear protein because of high fluorescence intensity on cell nucleus.?2?The level of Cyclin D1 mRNA in 25 ng?mL^-1 and 150 ng?m L^-11 groups were not significantly different with the control group?P>0.05?,while 50 and 100 ng?mL^-1groups were significantly higher than other groups?P<0.05?;the difference of Cyclin D1 protein expression between 100 ng?mL^-1 group and the control group was not remarkable?P>0.05?,however,a significant difference existed between the 25,50,150 ng?m L^-1 groups and control group?P<0.05?;the relative expression of Cyclin D1mRNA and protein were all reached the highest levels when IGF-I was 50 ng?mL^-1,which were 1.65 and 1.20 times than the control group respectively.?3?The relative expression level of Cyclin D1 mRNA adding TGF-?1 5,12,24 h were significantly higher than control group?P<0.05?,and that of the 18 h group was significantly lower than other groups?P<0.05?.The level of Cyclin D1 protein in different time treatment groups of TGF-?1 were significantly higher than control group?P<0.05?.When adding different concentration TGF-?1 into SCs cultured in vitro,the expression of Cyclin D1 mRNA and protein were significantly higher than that in control group?P<0.05?.TGF-?1 enhanced the expression of Cyclin D1 in a time-and dose-dependent manner,its optimal time and concentration were 12 h and750 pg?m L^-11 respectively.In conclusion,the results suggested that Cyclin D1 and Cyclin E1 gene were highly conserved during evolutionary process;IGF-I and TGF-?1 could regulate the growth of SCs by promoting the expression of Cyclin D1.This study provided the experimental basis for regulating the development of SCs.